WO2025184186A1 - Excision of exon 53 for treatment of duchenne muscular dystrophy - Google Patents

Abstract

Compositions and methods for treating Duchenne Muscular Dystrophy (DMD) and excising exon 53 of the DMD gene are encompassed.

Description

EXCISION OF EXON 53 FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63/559,662 filed February 29, 2024, and U.S. Provisional Patent Application No. 63/750,143 filed January 27, 2025, both of which are incorporated by reference herein in their entireties for all purposes.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 21, 2024, is named 01245-0061-60US.xml and is 4,327,739 bytes in size.

INTRODUCTION AND SUMMARY

[0003] Muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Duchenne muscular dystrophy (DMD) is one of the most severe forms of MD that affects approximately 1 in 5000 boys and is characterized by progressive muscle weakness and premature death. Cardiomyopathy and heart failure are common, incurable and lethal features of DMD. The disease is caused by mutations in the gene encoding dystrophin DMD), which result in loss of expression of dystrophin, causing muscle membrane fragility and progressive muscle wasting.

[0004] CRISPR-based genome editing can provide sequence-specific cleavage of genomic DNA using a Cas9 and a guide RNA. For example, a nucleic acid encoding the Cas9 enzyme and a nucleic acid encoding for the appropriate guide RNA can be provided on separate vectors or together on a single vector and administered in vivo or in vitro to knockout or correct a genetic mutation, for example. The approximately 20 nucleotides at the 5' end of the guide RNA serves as the guide or spacer sequence that can be any sequence complementary to one strand of a genomic target location that has an adjacent protospacer adjacent motif (PAM). The PAM sequence is a short sequence adjacent to the Cas9 nuclease cut site that the Cas9 molecule requires for appropriate binding. The nucleotides 3’ of the guide or spacer sequence of the guide RNA serve as a scaffold sequence for interacting with Cas9. When a guide RNA and a Cas9 are expressed, the guide RNA will bind to Cas9 and direct it to the sequence complementary to the guide sequence, where it will then initiate a double-stranded break (DSB). To repair these breaks, cells typically use the mechanism of non- homologous end joining (NHEJ). See, e.g., Kumar et al. (2018) Front. Mol. Neurosci. Vol. 11, Article 413.

[0005] While gene editing strategies using systems (e.g., CRISPR) for treating DMD have been previously explored, these strategies have focused primarily on either: a) cutting at multiple different sites to excise large portions (e.g., one or more exons) of the dystrophin gene (see, e.g., Ousterout et al., 2015, Nat Commun. 6:6244) using Streptococcus pyogenes Cas9 protein; or b) cutting in a single site to introduce indels that either result in a frame-shifting mutation and/or that destroy a splice acceptor/donor site in the dystrophin gene (see, e.g., Amoassi et al., 2018, Science, 362(6410):86-91). However, there remains a need for additional and alternative effective gene editing strategies for treating diseases like DMD.

[0006] Provided herein are compositions and methods for treating DMD utilizing Cas proteins such as Staphylococcus aureus (SaCas9) and Staphylococcus lugdunensis (SluCas9). In some embodiments, pairs of guide RNAs that excise all or a portion of exon 53 of the DMD gene are provided. In some embodiments, such a pair of guide RNAs is encoded on a single nucleic acid molecule.

[0007] Accordingly, the following non-limiting embodiments are provided.

[0008] Embodiment 1 is a composition comprising one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise i) a guide sequence of any one of the guide sequences shown in Tables 1A or 1C; ii) at least 16, 17, 18, 19, or 20 contiguous nucleotides of any one of the guide sequences shown in Tables 1A or 1C; iii) a guide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the guide sequences shown in Tables 1A or 1C; or iv) a pair of guide RNAs comprising any of the guide sequence pairs shown in Table 1D, optionally further comprising a Staphylococcus lugdunensis Cas9 (SluCas9) or a nucleic acid encoding a SluCas9.

[0009] Embodiment 2 is a composition comprising a first guide RNA and a second guide RNA, wherein a. the first guide RNA is selected from any one of SEQ ID NOs: 1-64; and b. the second guide RNA is selected from any one of SEQ ID NOs: 88-143.

[0010] Embodiment 3 is a composition comprising at least one pair of guide RNAs or a nucleic acid encoding at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprise or consist of a first and a second guide sequence selected from any of the following pairs of first and second guide sequences: a. SEQ ID NOs: 88 and 1; 88 and 2; 88 and 3; 88 and 4; 88 and 5; 88 and 6; 88 and 7; 88 and 8; 88 and 9; 88 and 10; 88 and 11; 88 and 12; 88 and 13; 88 and 14; 88 and 15; 88 and 16; 88 and 17; 88 and 18; 88 and 19; 88 and 20; 88 and 21; 88 and 22; 88 and 23; 88 and 24; 88 and 25; 88 and 26; 88 and 27; 88 and 28; 88 and 29; 88 and 30; 88 and 31; 88 and 32; 88 and 33; 88 and 34; 88 and 35; 88 and 36; 88 and 37; 88 and 38; 88 and 39; 88 and 40; 88 and 41; 88 and 42; 88 and 43; 88 and 44; 88 and 45; 88 and 46; 88 and 47; 88 and 48; 88 and 49; 88 and 50; 88 and 51; 88 and 52; 88 and 53; 88 and 54; 88 and 55; 88 and 56; 88 and 57; 88 and 58; 88 and 59; 88 and 60; 88 and 61; 88 and 62; 88 and 63; 88 and 64; 89 and 1; 89 and 2; 89 and 3; 89 and 4; 89 and 5; 89 and 6; 89 and 7; 89 and 8; 89 and 9; 89 and 10; 89 and 11; 89 and 12; 89 and 13; 89 and 14; 89 and 15; 89 and 16; 89 and 17; 89 and 18; 89 and 19; 89 and 20; 89 and 21; 89 and 22 ; 89 and 23; 89 and 24; 89 and 25; 89 and 26; 89 and 27; 89 and 28; 89 and 29; 89 and 30; 89 and 31 ; 89 and 32; 89 and 33; 89 and 34; 89 and 35; 89 and 36; 89 and 37; 89 and 38; 89 and 39; 89 and 40 ; 89 and 41; 89 and 42; 89 and 43; 89 and 44; 89 and 45; 89 and 46; 89 and 47; 89 and 48; 89 and 49 ; 89 and 50; 89 and 51; 89 and 52; 89 and 53; 89 and 54; 89 and 55; 89 and 56; 89 and 57; 89 and 58 ; 89 and 59; 89 and 60; 89 and 61; 89 and 62; 89 and 63; 89 and 64; 90 and 1; 90 and 2; 90 and 3; 90 and 4; 90 and 5; 90 and 6; 90 and 7; 90 and 8; 90 and 9; 90 and 10; 90 and 11; 90 and 12; 90 and 13; 90 and 14; 90 and 15; 90 and 16; 90 and 17; 90 and 18; 90 and 19; 90 and 20; 90 and 21; 90 and 22 ; 90 and 23; 90 and 24; 90 and 25; 90 and 26; 90 and 27; 90 and 28; 90 and 29; 90 and 30; 90 and 31 ; 90 and 32; 90 and 33; 90 and 34; 90 and 35; 90 and 36; 90 and 37; 90 and 38; 90 and 39; 90 and 40 ; 90 and 41; 90 and 42; 90 and 43; 90 and 44; 90 and 45; 90 and 46; 90 and 47; 90 and 48; 90 and 49 ; 90 and 50; 90 and 51; 90 and 52; 90 and 53; 90 and 54; 90 and 55; 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104 and 46; 104 and 47; 104 and 48; 104 and 49; 104 and 50; 104 and 51; 104 and 52; 104 and 53;

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107 and 26; 107 and 27; 107 and 28; 107 and 29; 107 and 30; 107 and 31; 107 and 32; 107 and 33;

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107 and 42; 107 and 43; 107 and 44; 107 and 45; 107 and 46; 107 and 47; 107 and 48; 107 and 49;

107 and 50; 107 and 51; 107 and 52; 107 and 53; 107 and 54; 107 and 55; 107 and 56; 107 and 57;

107 and 58; 107 and 59; 107 and 60; 107 and 61; 107 and 62; 107 and 63; 107 and 64; 108 and 1; 108 and 2; 108 and 3; 108 and 4; 108 and 5; 108 and 6; 108 and 7; 108 and 8; 108 and 9; 108 and 10; 108 and 11; 108 and 12; 108 and 13; 108 and 14; 108 and 15; 108 and 16; 108 and 17; 108 and 18; 108 and 19; 108 and 20; 108 and 21; 108 and 22; 108 and 23; 108 and 24; 108 and 25; 108 and 26; 108 and 27; 108 and 28; 108 and 29; 108 and 30; 108 and 31; 108 and 32; 108 and 33; 108 and 34; 108 and 35; 108 and 36; 108 and 37; 108 and 38; 108 and 39; 108 and 40; 108 and 41; 108 and 42; 108 and 43; 108 and 44; 108 and 45; 108 and 46; 108 and 47; 108 and 48; 108 and 49; 108 and 50; 108 and 51; 108 and 52; 108 and 53; 108 and 54; 108 and 55; 108 and 56; 108 and 57; 108 and 58; 108 and 59; 108 and 60; 108 and 61; 108 and 62; 108 and 63; 108 and 64; 109 and 1; 109 and 2; 109 and 3; 109 and 4; 109 and 5; 109 and 6; 109 and 7; 109 and 8; 109 and 9; 109 and 10; 109 and 11; 109 and 12; 109 and 13; 109 and 14; 109 and 15; 109 and 16; 109 and 17; 109 and 18; 109 and 19; 109 and 20; 109 and 21; 109 and 22; 109 and 23; 109 and 24; 109 and 25; 109 and 26; 109 and 27; 109 and

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110 and 46; 110 and 47; 110 and 48; 110 and 49; 110 and 50; 110 and 51; 110 and 52; 110 and 53;

110 and 54; 110 and 55; 110 and 56; 110 and 57; 110 and 58; 110 and 59; 110 and 60; 110 and 61;

110 and 62; 110 and 63; 110 and 64; 111 and 1; 111 and 2; 111 and 3; 111 and 4; 111 and 5; 111 and 6; 111 and 7; 111 and 8; 111 and 9; 111 and 10; 111 and 11; 111 and 12; 111 and 13; 111 and 14; 111 and 15; 111 and 16; 111 and 17; 111 and 18; 111 and 19; 111 and 20; 111 and 21; 111 and 22; 111 and 23; 111 and 24; 111 and 25; 111 and 26; 111 and 27; 111 and 28; 111 and 29; 111 and 30; 111 and 31; 111 and 32; 111 and 33; 111 and 34; 111 and 35; 111 and 36; 111 and 37; 111 and 38; 111 and 39; 111 and 40; 111 and 41; 111 and 42; 111 and 43; 111 and 44; 111 and 45; 111 and 46; 111 and 47; 111 and 48; 111 and 49; 111 and 50; 111 and 51; 111 and 52; 111 and 53; 111 and 54; 111 and 55; 111 and 56; 111 and 57; 111 and 58; 111 and 59; 111 and 60; 111 and 61; 111 and 62; 111 and 63; 111 and 64; 112 and 1; 112 and 2; 112 and 3; 112 and 4; 112 and 5; 112 and 6; 112 and 7; 112 and 8; 112 and 9; 112 and 10; 112 and 11; 112 and 12; 112 and 13; 112 and 14; 112 and 15; 112 and 16; 112 and 17; 112 and 18; 112 and 19; 112 and 20; 112 and 21; 112 and 22; 112 and 23; 112 and

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64; 113 and 1; 113 and 2; 113 and 3; 113 and 4; 113 and 5; 113 and 6; 113 and 7; 113 and 8; 113 and 9; 113 and 10; 113 and 11; 113 and 12; 113 and 13; 113 and 14; 113 and 15; 113 and 16; 113 and 17; 113 and 18; 113 and 19; 113 and 20; 113 and 21; 113 and 22; 113 and 23; 113 and 24; 113 and 25;

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113 and 42; 113 and 43; 113 and 44; 113 and 45; 113 and 46; 113 and 47; 113 and 48; 113 and 49;

113 and 50; 113 and 51; 113 and 52; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 57;

113 and 58; 113 and 59; 113 and 60; 113 and 61; 113 and 62; 113 and 63; 113 and 64; 114 and 1; 114 and 2; 114 and 3; 114 and 4; 114 and 5; 114 and 6; 114 and 7; 114 and 8; 114 and 9; 114 and 10; 114 and 11; 114 and 12; 114 and 13; 114 and 14; 114 and 15; 114 and 16; 114 and 17; 114 and 18; 114 and 19; 114 and 20; 114 and 21; 114 and 22; 114 and 23; 114 and 24; 114 and 25; 114 and 26; 114 and 27; 114 and 28; 114 and 29; 114 and 30; 114 and 31; 114 and 32; 114 and 33; 114 and 34; 114 and 35; 114 and 36; 114 and 37; 114 and 38; 114 and 39; 114 and 40; 114 and 41; 114 and 42; 114 and 43; 114 and 44; 114 and 45; 114 and 46; 114 and 47; 114 and 48; 114 and 49; 114 and 50; 114 and 51; 114 and 52; 114 and 53; 114 and 54; 114 and 55; 114 and 56; 114 and 57; 114 and 58; 114 and 59; 114 and 60; 114 and 61; 114 and 62; 114 and 63; 114 and 64; 115 and 1; 115 and 2; 115 and 3; 115 and 4; 115 and 5; 115 and 6; 115 and 7; 115 and 8; 115 and 9; 115 and 10; 115 and 11; 115 and 12; 115 and 13; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 18; 115 and 19; 115 and

20; 115 and 21; 115 and 22; 115 and 23; 115 and 24; 115 and 25; 115 and 26; 115 and 27; 115 and

28; 115 and 29; 115 and 30; 115 and 31; 115 and 32; 115 and 33; 115 and 34; 115 and 35; 115 and

36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 43; 115 and

44; 115 and 45; 115 and 46; 115 and 47; 115 and 48; 115 and 49; 115 and 50; 115 and 51; 115 and

52; 115 and 53; 115 and 54; 115 and 55; 115 and 56; 115 and 57; 115 and 58; 115 and 59; 115 and

60; 115 and 61; 115 and 62; 115 and 63; 115 and 64; 116 and 1; 116 and 2; 116 and 3; 116 and 4; 116 and 5; 116 and 6; 116 and 7; 116 and 8; 116 and 9; 116 and 10; 116 and 11; 116 and 12; 116 and 13; 116 and 14; 116 and 15; 116 and 16; 116 and 17; 116 and 18; 116 and 19; 116 and 20; 116 and 21;

116 and 22; 116 and 23; 116 and 24; 116 and 25; 116 and 26; 116 and 27; 116 and 28; 116 and 29;

116 and 30; 116 and 31; 116 and 32; 116 and 33; 116 and 34; 116 and 35; 116 and 36; 116 and 37;

116 and 38; 116 and 39; 116 and 40; 116 and 41; 116 and 42; 116 and 43; 116 and 44; 116 and 45;

116 and 46; 116 and 47; 116 and 48; 116 and 49; 116 and 50; 116 and 51; 116 and 52; 116 and 53;

116 and 54; 116 and 55; 116 and 56; 116 and 57; 116 and 58; 116 and 59; 116 and 60; 116 and 61;

116 and 62; 116 and 63; 116 and 64; 117 and 1; 117 and 2; 117 and 3; 117 and 4; 117 and 5; 117 and 6; 117 and 7; 117 and 8; 117 and 9; 117 and 10; 117 and 11; 117 and 12; 117 and 13; 117 and 14; 117 and 15; 117 and 16; 117 and 17; 117 and 18; 117 and 19; 117 and 20; 117 and 21; 117 and 22; 117 and 23; 117 and 24; 117 and 25; 117 and 26; 117 and 27; 117 and 28; 117 and 29; 117 and 30; 117 and 31; 117 and 32; 117 and 33; 117 and 34; 117 and 35; 117 and 36; 117 and 37; 117 and 38; 117 and 39; 117 and 40; 117 and 41; 117 and 42; 117 and 43; 117 and 44; 117 and 45; 117 and 46; 117 and 47; 117 and 48; 117 and 49; 117 and 50; 117 and 51; 117 and 52; 117 and 53; 117 and 54; 117 and 55; 117 and 56; 117 and 57; 117 and 58; 117 and 59; 117 and 60; 117 and 61; 117 and 62; 117 and 63; 117 and 64; 118 and 1; 118 and 2; 118 and 3; 118 and 4; 118 and 5; 118 and 6; 118 and 7; 118 and 8; 118 and 9; 118 and 10; 118 and 11; 118 and 12; 118 and 13; 118 and 14; 118 and 15; 118 and 16; 118 and 17; 118 and 18; 118 and 19; 118 and 20; 118 and 21; 118 and 22; 118 and 23; 118 and

24; 118 and 25; 118 and 26; 118 and 27; 118 and 28; 118 and 29; 118 and 30; 118 and 31; 118 and

32; 118 and 33; 118 and 34; 118 and 35; 118 and 36; 118 and 37; 118 and 38; 118 and 39; 118 and

40; 118 and 41; 118 and 42; 118 and 43; 118 and 44; 118 and 45; 118 and 46; 118 and 47; 118 and 48; 118 and 49; 118 and 50; 118 and 51; 118 and 52; 118 and 53; 118 and 54; 118 and 55; 118 and 56; 118 and 57; 118 and 58; 118 and 59; 118 and 60; 118 and 61; 118 and 62; 118 and 63; 118 and 64; 119 and 1; 119 and 2; 119 and 3; 119 and 4; 119 and 5; 119 and 6; 119 and 7; 119 and 8; 119 and 9; 119 and 10; 119 and 11; 119 and 12; 119 and 13; 119 and 14; 119 and 15; 119 and 16; 119 and 17; 119 and 18; 119 and 19; 119 and 20; 119 and 21; 119 and 22; 119 and 23; 119 and 24; 119 and 25; 119 and 26; 119 and 27; 119 and 28; 119 and 29; 119 and 30; 119 and 31; 119 and 32; 119 and 33; 119 and 34; 119 and 35; 119 and 36; 119 and 37; 119 and 38; 119 and 39; 119 and 40; 119 and 41; 119 and 42; 119 and 43; 119 and 44; 119 and 45; 119 and 46; 119 and 47; 119 and 48; 119 and 49; 119 and 50; 119 and 51; 119 and 52; 119 and 53; 119 and 54; 119 and 55; 119 and 56; 119 and 57; 119 and 58; 119 and 59; 119 and 60; 119 and 61; 119 and 62; 119 and 63; 119 and 64; 120 and l; 120 and 2; 120 and 3; 120 and 4; 120 and 5; 120 and 6; 120 and 7; 120 and 8; 120 and 9; 120 and 10; 120 and 11; 120 and 12; 120 and 13; 120 and 14; 120 and 15; 120 and 16; 120 and 17; 120 and 18; 120 and 19; 120 and 20; 120 and 21; 120 and 22; 120 and 23; 120 and 24; 120 and 25; 120 and 26; 120 and 27; 120 and 28; 120 and 29; 120 and 30; 120 and 31; 120 and 32; 120 and 33; 120 and 34; 120 and 35; 120 and 36; 120 and 37; 120 and 38; 120 and 39; 120 and 40; 120 and 41; 120 and 42; 120 and 43; 120 and 44; 120 and 45; 120 and 46; 120 and 47; 120 and 48; 120 and 49; 120 and 50; 120 and 51; 120 and 52; 120 and 53; 120 and 54; 120 and 55; 120 and 56; 120 and 57; 120 and 58; 120 and 59; 120 and 60; 120 and 61; 120 and 62; 120 and 63; 120 and 64; 121 and 1; 121 and 2; 121 and 3; 121 and 4; 121 and 5; 121 and 6; 121 and 7; 121 and 8; 121 and 9; 121 and 10; 121 and 11; 121 and 12; 121 and 13; 121 and 14; 121 and 15; 121 and 16; 121 and 17; 121 and 18; 121 and 19; 121 and

20; 121 and 21; 121 and 22; 121 and 23; 121 and 24; 121 and 25; 121 and 26; 121 and 27; 121 and

28; 121 and 29; 121 and 30; 121 and 31; 121 and 32; 121 and 33; 121 and 34; 121 and 35; 121 and

36; 121 and 37; 121 and 38; 121 and 39; 121 and 40; 121 and 41; 121 and 42; 121 and 43; 121 and

44; 121 and 45; 121 and 46; 121 and 47; 121 and 48; 121 and 49; 121 and 50; 121 and 51; 121 and

52; 121 and 53; 121 and 54; 121 and 55; 121 and 56; 121 and 57; 121 and 58; 121 and 59; 121 and

60; 121 and 61; 121 and 62; 121 and 63; 121 and 64; 122 and 1; 122 and 2; 122 and 3; 122 and 4; 122 and 5; 122 and 6; 122 and 7; 122 and 8; 122 and 9; 122 and 10; 122 and 11; 122 and 12; 122 and 13; 122 and 14; 122 and 15; 122 and 16; 122 and 17; 122 and 18; 122 and 19; 122 and 20; 122 and 21;

122 and 22; 122 and 23; 122 and 24; 122 and 25; 122 and 26; 122 and 27; 122 and 28; 122 and 29;

122 and 30; 122 and 31; 122 and 32; 122 and 33; 122 and 34; 122 and 35; 122 and 36; 122 and 37;

122 and 38; 122 and 39; 122 and 40; 122 and 41; 122 and 42; 122 and 43; 122 and 44; 122 and 45;

122 and 46; 122 and 47; 122 and 48; 122 and 49; 122 and 50; 122 and 51; 122 and 52; 122 and 53;

122 and 54; 122 and 55; 122 and 56; 122 and 57; 122 and 58; 122 and 59; 122 and 60; 122 and 61;

122 and 62; 122 and 63; 122 and 64; 123 and 1; 123 and 2; 123 and 3; 123 and 4; 123 and 5; 123 and 6; 123 and 7; 123 and 8; 123 and 9; 123 and 10; 123 and 11; 123 and 12; 123 and 13; 123 and 14; 123 and 15; 123 and 16; 123 and 17; 123 and 18; 123 and 19; 123 and 20; 123 and 21; 123 and 22; 123 and 23; 123 and 24; 123 and 25; 123 and 26; 123 and 27; 123 and 28; 123 and 29; 123 and 30; 123 and 31; 123 and 32; 123 and 33; 123 and 34; 123 and 35; 123 and 36; 123 and 37; 123 and 38; 123 and 39; 123 and 40; 123 and 41; 123 and 42; 123 and 43; 123 and 44; 123 and 45; 123 and 46; 123 and 47; 123 and 48; 123 and 49; 123 and 50; 123 and 51; 123 and 52; 123 and 53; 123 and 54; 123 and 55; 123 and 56; 123 and 57; 123 and 58; 123 and 59; 123 and 60; 123 and 61; 123 and 62; 123 and 63; 123 and 64; 124 and 1; 124 and 2; 124 and 3; 124 and 4; 124 and 5; 124 and 6; 124 and 7; 124 and 8; 124 and 9; 124 and 10; 124 and 11; 124 and 12; 124 and 13; 124 and 14; 124 and 15; 124 and 16; 124 and 17; 124 and 18; 124 and 19; 124 and 20; 124 and 21; 124 and 22; 124 and 23; 124 and 24; 124 and 25; 124 and 26; 124 and 27; 124 and 28; 124 and 29; 124 and 30; 124 and 31; 124 and 32; 124 and 33; 124 and 34; 124 and 35; 124 and 36; 124 and 37; 124 and 38; 124 and 39; 124 and 40; 124 and 41; 124 and 42; 124 and 43; 124 and 44; 124 and 45; 124 and 46; 124 and 47; 124 and 48; 124 and 49; 124 and 50; 124 and 51; 124 and 52; 124 and 53; 124 and 54; 124 and 55; 124 and 56; 124 and 57; 124 and 58; 124 and 59; 124 and 60; 124 and 61; 124 and 62; 124 and 63; 124 and 64; 125 and 1; 125 and 2; 125 and 3; 125 and 4; 125 and 5; 125 and 6; 125 and 7; 125 and 8; 125 and 9; 125 and 10; 125 and 11; 125 and 12; 125 and 13; 125 and 14; 125 and 15; 125 and 16; 125 and 17; 125 and 18; 125 and 19; 125 and 20; 125 and 21; 125 and 22; 125 and 23; 125 and 24; 125 and 25; 125 and 26; 125 and 27; 125 and 28; 125 and 29; 125 and 30; 125 and 31; 125 and 32; 125 and 33; 125 and 34; 125 and 35; 125 and 36; 125 and 37; 125 and 38; 125 and 39; 125 and 40; 125 and 41; 125 and 42; 125 and 43; 125 and 44; 125 and 45; 125 and 46; 125 and 47; 125 and 48; 125 and 49; 125 and 50; 125 and 51; 125 and 52; 125 and 53; 125 and 54; 125 and 55; 125 and 56; 125 and 57; 125 and 58; 125 and 59; 125 and 60; 125 and 61; 125 and 62; 125 and 63; 125 and 64; 126 and 1; 126 and 2; 126 and 3; 126 and 4; 126 and 5; 126 and 6; 126 and 7; 126 and 8; 126 and 9; 126 and 10; 126 and 11; 126 and 12; 126 and 13; 126 and 14; 126 and 15; 126 and 16; 126 and 17; 126 and 18; 126 and 19; 126 and 20; 126 and 21; 126 and 22; 126 and 23; 126 and 24; 126 and 25; 126 and 26; 126 and 27; 126 and 28; 126 and 29; 126 and 30; 126 and 31; 126 and 32; 126 and 33; 126 and 34; 126 and 35; 126 and 36; 126 and 37; 126 and 38; 126 and 39; 126 and 40; 126 and 41; 126 and 42; 126 and 43; 126 and 44; 126 and 45; 126 and 46; 126 and 47; 126 and 48; 126 and 49; 126 and 50; 126 and 51; 126 and 52; 126 and 53; 126 and 54; 126 and 55; 126 and 56; 126 and 57; 126 and 58; 126 and 59; 126 and 60; 126 and 61; 126 and 62; 126 and 63; 126 and 64; 127 and 1; 127 and 2; 127 and 3; 127 and 4; 127 and 5; 127 and 6; 127 and 7; 127 and 8; 127 and 9; 127 and 10; 127 and 11; 127 and 12; 127 and 13; 127 and 14; 127 and 15; 127 and 16; 127 and 17; 127 and 18; 127 and 19; 127 and

20; 127 and 21; 127 and 22; 127 and 23; 127 and 24; 127 and 25; 127 and 26; 127 and 27; 127 and

28; 127 and 29; 127 and 30; 127 and 31; 127 and 32; 127 and 33; 127 and 34; 127 and 35; 127 and

36; 127 and 37; 127 and 38; 127 and 39; 127 and 40; 127 and 41; 127 and 42; 127 and 43; 127 and

44; 127 and 45; 127 and 46; 127 and 47; 127 and 48; 127 and 49; 127 and 50; 127 and 51; 127 and

52; 127 and 53; 127 and 54; 127 and 55; 127 and 56; 127 and 57; 127 and 58; 127 and 59; 127 and

60; 127 and 61; 127 and 62; 127 and 63; 127 and 64; 128 and 1; 128 and 2; 128 and 3; 128 and 4; 128 and 5; 128 and 6; 128 and 7; 128 and 8; 128 and 9; 128 and 10; 128 and 11; 128 and 12; 128 and 13; 128 and 14; 128 and 15; 128 and 16; 128 and 17; 128 and 18; 128 and 19; 128 and 20; 128 and 21;

128 and 22; 128 and 23; 128 and 24; 128 and 25; 128 and 26; 128 and 27; 128 and 28; 128 and 29;

128 and 30; 128 and 31; 128 and 32; 128 and 33; 128 and 34; 128 and 35; 128 and 36; 128 and 37;

128 and 38; 128 and 39; 128 and 40; 128 and 41; 128 and 42; 128 and 43; 128 and 44; 128 and 45;

128 and 46; 128 and 47; 128 and 48; 128 and 49; 128 and 50; 128 and 51; 128 and 52; 128 and 53;

128 and 54; 128 and 55; 128 and 56; 128 and 57; 128 and 58; 128 and 59; 128 and 60; 128 and 61;

128 and 62; 128 and 63; 128 and 64; 129 and 1; 129 and 2; 129 and 3; 129 and 4; 129 and 5; 129 and 6; 129 and 7; 129 and 8; 129 and 9; 129 and 10; 129 and 11; 129 and 12; 129 and 13; 129 and 14; 129 and 15; 129 and 16; 129 and 17; 129 and 18; 129 and 19; 129 and 20; 129 and 21; 129 and 22; 129 and 23; 129 and 24; 129 and 25; 129 and 26; 129 and 27; 129 and 28; 129 and 29; 129 and 30; 129 and 31; 129 and 32; 129 and 33; 129 and 34; 129 and 35; 129 and 36; 129 and 37; 129 and 38; 129 and 39; 129 and 40; 129 and 41; 129 and 42; 129 and 43; 129 and 44; 129 and 45; 129 and 46; 129 and 47; 129 and 48; 129 and 49; 129 and 50; 129 and 51; 129 and 52; 129 and 53; 129 and 54; 129 and 55; 129 and 56; 129 and 57; 129 and 58; 129 and 59; 129 and 60; 129 and 61; 129 and 62; 129 and 63; 129 and 64; 130 and 1; 130 and 2; 130 and 3; 130 and 4; 130 and 5; 130 and 6; 130 and 7; 130 and 8; 130 and 9; 130 and 10; 130 and 11; 130 and 12; 130 and 13; 130 and 14; 130 and 15; 130 and 16; 130 and 17; 130 and 18; 130 and 19; 130 and 20; 130 and 21; 130 and 22; 130 and 23; 130 and

24; 130 and 25; 130 and 26; 130 and 27; 130 and 28; 130 and 29; 130 and 30; 130 and 31; 130 and

32; 130 and 33; 130 and 34; 130 and 35; 130 and 36; 130 and 37; 130 and 38; 130 and 39; 130 and

40; 130 and 41; 130 and 42; 130 and 43; 130 and 44; 130 and 45; 130 and 46; 130 and 47; 130 and

48; 130 and 49; 130 and 50; 130 and 51; 130 and 52; 130 and 53; 130 and 54; 130 and 55; 130 and

56; 130 and 57; 130 and 58; 130 and 59; 130 and 60; 130 and 61; 130 and 62; 130 and 63; 130 and

64; 131 and 1; 131 and 2; 131 and 3; 131 and 4; 131 and 5; 131 and 6; 131 and 7; 131 and 8; 131 and 9; 131 and 10; 131 and 11; 131 and 12; 131 and 13; 131 and 14; 131 and 15; 131 and 16; 131 and 17; 131 and 18; 131 and 19; 131 and 20; 131 and 21; 131 and 22; 131 and 23; 131 and 24; 131 and 25;

131 and 26; 131 and 27; 131 and 28; 131 and 29; 131 and 30; 131 and 31; 131 and 32; 131 and 33;

131 and 34; 131 and 35; 131 and 36; 131 and 37; 131 and 38; 131 and 39; 131 and 40; 131 and 41;

131 and 42; 131 and 43; 131 and 44; 131 and 45; 131 and 46; 131 and 47; 131 and 48; 131 and 49;

131 and 50; 131 and 51; 131 and 52; 131 and 53; 131 and 54; 131 and 55; 131 and 56; 131 and 57;

131 and 58; 131 and 59; 131 and 60; 131 and 61; 131 and 62; 131 and 63; 131 and 64; 132 and l; 132 and 2; 132 and 3; 132 and 4; 132 and 5; 132 and 6; 132 and 7; 132 and 8; 132 and 9; 132 and 10; 132 and 11; 132 and 12; 132 and 13; 132 and 14; 132 and 15; 132 and 16; 132 and 17; 132 and 18; 132 and 19; 132 and 20; 132 and 21; 132 and 22; 132 and 23; 132 and 24; 132 and 25; 132 and 26; 132 and 27; 132 and 28; 132 and 29; 132 and 30; 132 and 31; 132 and 32; 132 and 33; 132 and 34; 132 and 35; 132 and 36; 132 and 37; 132 and 38; 132 and 39; 132 and 40; 132 and 41; 132 and 42; 132 and 43; 132 and 44; 132 and 45; 132 and 46; 132 and 47; 132 and 48; 132 and 49; 132 and 50; 132 and 51; 132 and 52; 132 and 53; 132 and 54; 132 and 55; 132 and 56; 132 and 57; 132 and 58; 132 and 59; 132 and 60; 132 and 61; 132 and 62; 132 and 63; 132 and 64; 133 and 1; 133 and 2; 133 and 3; 133 and 4; 133 and 5; 133 and 6; 133 and 7; 133 and 8; 133 and 9; 133 and 10; 133 and 11; 133 and 12; 133 and 13; 133 and 14; 133 and 15; 133 and 16; 133 and 17; 133 and 18; 133 and 19; 133 and

20; 133 and 21; 133 and 22; 133 and 23; 133 and 24; 133 and 25; 133 and 26; 133 and 27; 133 and

28; 133 and 29; 133 and 30; 133 and 31; 133 and 32; 133 and 33; 133 and 34; 133 and 35; 133 and

36; 133 and 37; 133 and 38; 133 and 39; 133 and 40; 133 and 41; 133 and 42; 133 and 43; 133 and

44; 133 and 45; 133 and 46; 133 and 47; 133 and 48; 133 and 49; 133 and 50; 133 and 51; 133 and

52; 133 and 53; 133 and 54; 133 and 55; 133 and 56; 133 and 57; 133 and 58; 133 and 59; 133 and

60; 133 and 61; 133 and 62; 133 and 63; 133 and 64; 134 and 1; 134 and 2; 134 and 3; 134 and 4; 134 and 5; 134 and 6; 134 and 7; 134 and 8; 134 and 9; 134 and 10; 134 and 11; 134 and 12; 134 and 13; 134 and 14; 134 and 15; 134 and 16; 134 and 17; 134 and 18; 134 and 19; 134 and 20; 134 and 21;

134 and 22; 134 and 23; 134 and 24; 134 and 25; 134 and 26; 134 and 27; 134 and 28; 134 and 29;

134 and 30; 134 and 31; 134 and 32; 134 and 33; 134 and 34; 134 and 35; 134 and 36; 134 and 37;

134 and 38; 134 and 39; 134 and 40; 134 and 41; 134 and 42; 134 and 43; 134 and 44; 134 and 45;

134 and 46; 134 and 47; 134 and 48; 134 and 49; 134 and 50; 134 and 51; 134 and 52; 134 and 53;

134 and 54; 134 and 55; 134 and 56; 134 and 57; 134 and 58; 134 and 59; 134 and 60; 134 and 61;

134 and 62; 134 and 63; 134 and 64; 135 and 1; 135 and 2; 135 and 3; 135 and 4; 135 and 5; 135 and 6; 135 and 7; 135 and 8; 135 and 9; 135 and 10; 135 and 11; 135 and 12; 135 and 13; 135 and 14; 135 and 15; 135 and 16; 135 and 17; 135 and 18; 135 and 19; 135 and 20; 135 and 21; 135 and 22; 135 and 23; 135 and 24; 135 and 25; 135 and 26; 135 and 27; 135 and 28; 135 and 29; 135 and 30; 135 and 31; 135 and 32; 135 and 33; 135 and 34; 135 and 35; 135 and 36; 135 and 37; 135 and 38; 135 and 39; 135 and 40; 135 and 41; 135 and 42; 135 and 43; 135 and 44; 135 and 45; 135 and 46; 135 and 47; 135 and 48; 135 and 49; 135 and 50; 135 and 51; 135 and 52; 135 and 53; 135 and 54; 135 and 55; 135 and 56; 135 and 57; 135 and 58; 135 and 59; 135 and 60; 135 and 61; 135 and 62; 135 and 63; 135 and 64; 136 and 1; 136 and 2; 136 and 3; 136 and 4; 136 and 5; 136 and 6; 136 and 7; 136 and 8; 136 and 9; 136 and 10; 136 and 11; 136 and 12; 136 and 13; 136 and 14; 136 and 15; 136 and 16; 136 and 17; 136 and 18; 136 and 19; 136 and 20; 136 and 21; 136 and 22; 136 and 23; 136 and

24; 136 and 25; 136 and 26; 136 and 27; 136 and 28; 136 and 29; 136 and 30; 136 and 31; 136 and

32; 136 and 33; 136 and 34; 136 and 35; 136 and 36; 136 and 37; 136 and 38; 136 and 39; 136 and

40; 136 and 41; 136 and 42; 136 and 43; 136 and 44; 136 and 45; 136 and 46; 136 and 47; 136 and

48; 136 and 49; 136 and 50; 136 and 51; 136 and 52; 136 and 53; 136 and 54; 136 and 55; 136 and

56; 136 and 57; 136 and 58; 136 and 59; 136 and 60; 136 and 61; 136 and 62; 136 and 63; 136 and

64; 137 and 1; 137 and 2; 137 and 3; 137 and 4; 137 and 5; 137 and 6; 137 and 7; 137 and 8; 137 and 9; 137 and 10; 137 and 11; 137 and 12; 137 and 13; 137 and 14; 137 and 15; 137 and 16; 137 and 17;

137 and 18; 137 and 19; 137 and 20; 137 and 21; 137 and 22; 137 and 23; 137 and 24; 137 and 25;

137 and 26; 137 and 27; 137 and 28; 137 and 29; 137 and 30; 137 and 31; 137 and 32; 137 and 33;

137 and 34; 137 and 35; 137 and 36; 137 and 37; 137 and 38; 137 and 39; 137 and 40; 137 and 41; 137 and 42; 137 and 43; 137 and 44; 137 and 45; 137 and 46; 137 and 47; 137 and 48; 137 and 49;

137 and 50; 137 and 51; 137 and 52; 137 and 53; 137 and 54; 137 and 55; 137 and 56; 137 and 57;

137 and 58; 137 and 59; 137 and 60; 137 and 61; 137 and 62; 137 and 63; 137 and 64; 138 and 1; 138 and 2; 138 and 3; 138 and 4; 138 and 5; 138 and 6; 138 and 7; 138 and 8; 138 and 9; 138 and 10; 138 and 11; 138 and 12; 138 and 13; 138 and 14; 138 and 15; 138 and 16; 138 and 17; 138 and 18; 138 and 19; 138 and 20; 138 and 21; 138 and 22; 138 and 23; 138 and 24; 138 and 25; 138 and 26; 138 and 27; 138 and 28; 138 and 29; 138 and 30; 138 and 31; 138 and 32; 138 and 33; 138 and 34; 138 and 35; 138 and 36; 138 and 37; 138 and 38; 138 and 39; 138 and 40; 138 and 41; 138 and 42; 138 and 43; 138 and 44; 138 and 45; 138 and 46; 138 and 47; 138 and 48; 138 and 49; 138 and 50; 138 and 51; 138 and 52; 138 and 53; 138 and 54; 138 and 55; 138 and 56; 138 and 57; 138 and 58; 138 and 59; 138 and 60; 138 and 61; 138 and 62; 138 and 63; 138 and 64; 139 and 1; 139 and 2; 139 and 3; 139 and 4; 139 and 5; 139 and 6; 139 and 7; 139 and 8; 139 and 9; 139 and 10; 139 and 11; 139 and 12; 139 and 13; 139 and 14; 139 and 15; 139 and 16; 139 and 17; 139 and 18; 139 and 19; 139 and

20; 139 and 21; 139 and 22; 139 and 23; 139 and 24; 139 and 25; 139 and 26; 139 and 27; 139 and

28; 139 and 29; 139 and 30; 139 and 31; 139 and 32; 139 and 33; 139 and 34; 139 and 35; 139 and

36; 139 and 37; 139 and 38; 139 and 39; 139 and 40; 139 and 41; 139 and 42; 139 and 43; 139 and

44; 139 and 45; 139 and 46; 139 and 47; 139 and 48; 139 and 49; 139 and 50; 139 and 51; 139 and

52; 139 and 53; 139 and 54; 139 and 55; 139 and 56; 139 and 57; 139 and 58; 139 and 59; 139 and

60; 139 and 61; 139 and 62; 139 and 63; 139 and 64; 140 and 1; 140 and 2; 140 and 3; 140 and 4; 140 and 5; 140 and 6; 140 and 7; 140 and 8; 140 and 9; 140 and 10; 140 and 11; 140 and 12; 140 and 13; 140 and 14; 140 and 15; 140 and 16; 140 and 17; 140 and 18; 140 and 19; 140 and 20; 140 and 21;

140 and 22; 140 and 23; 140 and 24; 140 and 25; 140 and 26; 140 and 27; 140 and 28; 140 and 29;

140 and 30; 140 and 31; 140 and 32; 140 and 33; 140 and 34; 140 and 35; 140 and 36; 140 and 37;

140 and 38; 140 and 39; 140 and 40; 140 and 41; 140 and 42; 140 and 43; 140 and 44; 140 and 45;

140 and 46; 140 and 47; 140 and 48; 140 and 49; 140 and 50; 140 and 51; 140 and 52; 140 and 53;

140 and 54; 140 and 55; 140 and 56; 140 and 57; 140 and 58; 140 and 59; 140 and 60; 140 and 61;

140 and 62; 140 and 63; 140 and 64; 141 and 1; 141 and 2; 141 and 3; 141 and 4; 141 and 5; 141 and 6; 141 and 7; 141 and 8; 141 and 9; 141 and 10; 141 and 11; 141 and 12; 141 and 13; 141 and 14; 141 and 15; 141 and 16; 141 and 17; 141 and 18; 141 and 19; 141 and 20; 141 and 21; 141 and 22; 141 and 23; 141 and 24; 141 and 25; 141 and 26; 141 and 27; 141 and 28; 141 and 29; 141 and 30; 141 and 31; 141 and 32; 141 and 33; 141 and 34; 141 and 35; 141 and 36; 141 and 37; 141 and 38; 141 and 39; 141 and 40; 141 and 41; 141 and 42; 141 and 43; 141 and 44; 141 and 45; 141 and 46; 141 and 47; 141 and 48; 141 and 49; 141 and 50; 141 and 51; 141 and 52; 141 and 53; 141 and 54; 141 and 55; 141 and 56; 141 and 57; 141 and 58; 141 and 59; 141 and 60; 141 and 61; 141 and 62; 141 and 63; 141 and 64; 142 and 1; 142 and 2; 142 and 3; 142 and 4; 142 and 5; 142 and 6; 142 and 7; 142 and 8; 142 and 9; 142 and 10; 142 and 11; 142 and 12; 142 and 13; 142 and 14; 142 and 15; 142 and 16; 142 and 17; 142 and 18; 142 and 19; 142 and 20; 142 and 21; 142 and 22; 142 and 23; 142 and 24; 142 and 25; 142 and 26; 142 and 27; 142 and 28; 142 and 29; 142 and 30; 142 and 31; 142 and

32; 142 and 33; 142 and 34; 142 and 35; 142 and 36; 142 and 37; 142 and 38; 142 and 39; 142 and

40; 142 and 41; 142 and 42; 142 and 43; 142 and 44; 142 and 45; 142 and 46; 142 and 47; 142 and

48; 142 and 49; 142 and 50; 142 and 51; 142 and 52; 142 and 53; 142 and 54; 142 and 55; 142 and

56; 142 and 57; 142 and 58; 142 and 59; 142 and 60; 142 and 61; 142 and 62; 142 and 63; 142 and

64; 143 and 1; 143 and 2; 143 and 3; 143 and 4; 143 and 5; 143 and 6; 143 and 7; 143 and 8; 143 and 9; 143 and 10; 143 and 11; 143 and 12; 143 and 13; 143 and 14; 143 and 15; 143 and 16; 143 and 17; 143 and 18; 143 and 19; 143 and 20; 143 and 21; 143 and 22; 143 and 23; 143 and 24; 143 and 25;

143 and 26; 143 and 27; 143 and 28; 143 and 29; 143 and 30; 143 and 31; 143 and 32; 143 and 33;

143 and 34; 143 and 35; 143 and 36; 143 and 37; 143 and 38; 143 and 39; 143 and 40; 143 and 41;

143 and 42; 143 and 43; 143 and 44; 143 and 45; 143 and 46; 143 and 47; 143 and 48; 143 and 49;

143 and 50; 143 and 51; 143 and 52; 143 and 53; 143 and 54; 143 and 55; 143 and 56; 143 and 57;

143 and 58; 143 and 59; 143 and 60; 143 and 61; 143 and 62; 143 and 63; or 143 and 64; or b. SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28; 113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48;

113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and 16;

113 and 17; 113 and 57; 113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and 23;

113 and 24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48; 115 and 13; 115 and 53; 115 and 54; 115 and 55; 115 and 56; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18; 115 and 58; 115 and 19; 115 and 20; 115 and 59; 115 and 23; 115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and 26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42.

[0011] Embodiment 4 is a composition comprising one or more nucleic acid molecules encoding: a. a Staphylococcus lugdunensis Cas9 (SluCas9); and b. at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first guide sequence and a second guide sequence selected from any of the pairs of guide sequences shown in Table ID.

[0012] Embodiment 5 is a composition comprising: a. a single nucleic acid molecule comprising: i. a nucleic acid encoding Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; or ii. a nucleic acid encoding SluCas9 and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii. a nucleic acid encoding SluCas9 and 1, 2, or 3 guide RNAs; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A or 1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide sequence selected from the pairs of guide sequences shown in Table 1D, and further optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence; or b. two nucleic acid molecules comprising: i. a first nucleic acid encoding SluCas9; and a second nucleic acid that does not encode a SluCas9 and encodes any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; ii. a first nucleic acid encoding SluCas9 and

1. at least one, at least two, or at least three guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. 1, 2, or 3 guide RNAs; and a second nucleic acid that does not encode a SluCas9, optionally wherein the second nucleic acid comprises any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; iii. a first nucleic acid encoding SluCas9 and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SluCas9 and encodes from one to six guide RNAs; or iv. a first nucleic acid encoding SluCas9 and at least two guide RNAs, and a second nucleic acid that does not encode a SluCas9 and encodes at least one additional copy of each of the guide RNAs encoded in the first nucleic acid; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A or 1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from the pairs of guide sequences shown in Table 1D, and optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence.

[0013] Embodiment 6 is a composition comprising a single nucleic acid molecule encoding at least one pair of guide RNAs and a Cas9, wherein the single nucleic acid molecule comprises: a. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences shown in Table 1D; and a second nucleic acid encoding a Staphylococcus lugdunensis Cas9 (SluCas9); or b. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences of: SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28; 113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48; 113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and

16; 113 and 17; 113 and 57; 113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and

23; 113 and 24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45;

115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48; 115 and 13; 115 and 53; 115 and 54;

115 and 55; 115 and 56; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18;

115 and 58; 115 and 19; 115 and 20; 115 and 59; 115 and 23; 115 and 24; 115 and 60; 115 and 62;

115 and 63; 115 and 64; 115 and 25; 115 and 26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and l; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42; and a second nucleic acid encoding a SluCas9. [0014] Embodiment 7 is a composition comprising a first and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a Staphylococcus lugdunensis (SluCas9) endonuclease and optionally a first or a first and second guide RNA, and the second nucleic acid molecule comprises a first or a first and second guide RNA, wherein the first guide RNA comprises a first guide sequence and the second guide RNA comprises a second guide sequence selected from any one the following pairs of first and second guide sequences: a. any one of the pairs of first and second guide sequences shown in Table ID; or b. SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28; 113 and

35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48;

113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and 16;

113 and 17; 113 and 57; 113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and 23;

113 and 24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48; 115 and 13; 115 and 53; 115 and 54; 115 and 55; 115 and 56; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18; 115 and 58; 115 and 19; 115 and 20; 115 and 59; 115 and 23; 115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and 26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42.

[0015] Embodiment 8 is a composition comprising one or more nucleic acid molecules encoding a Staphylococcus lugdunensis Cas9 (SluCas9) and at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide RNA, and wherein the first guide RNA targets a sequence in intron 53 of a DMD gene and the second guide RNA targets a sequence in intron 52 of the DMD gene.

[0016] Embodiment 9 is the composition of any one of embodiments 1-8, comprising at least one pair of guide RNAs, wherein the at least one pair of guide RNAs is capable of excising at least a portion of exon 53 of the DMD gene.

[0017] Embodiment 10 is the composition of any one of embodiments 1-9, comprising at least one pair of guide RNAs, wherein the first and the second guide RNAs of the at least one pair of guide RNAs each comprise a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a first and a second guide sequence selected from any one of the following pairs of first and second guide sequences: a. any one of the pairs of first and second guide sequences shown in Table ID; or b. SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28; 113 and

35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48;

113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and 16;

113 and 17; 113 and 57; 113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and 23;

113 and 24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48; 115 and 13; 115 and 53; 115 and 54; 115 and 55; 115 and 56; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18; 115 and 58; 115 and 19; 115 and 20; 115 and 59; 115 and 23; 115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and 26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2;

120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1;

122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42.

[0018] Embodiment 11 is the composition of any one of embodiments 1-10, wherein: i) the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 2; ii) the first guide RNA comprises the sequence of SEQ ID NO: 115, and the second guide RNA comprises the sequence of SEQ ID NO: 3; or iii) the first guide RNA comprises the sequence of SEQ ID NO: 113, and the second guide RNA comprises the sequence of SEQ ID NO: 3.

[0019] Embodiment 12 is a composition comprising at least one pair of guide RNAs, wherein the at least one pair of guide RNAs is capable of excising a fragment of exon 53 of the DMD gene, and wherein i. the fragment comprises a splice donor site, a first guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases upstream of the splice donor site, and a second guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases downstream of the splice donor site; or ii. the fragment comprises a splice acceptor site, a first guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases upstream of the splice acceptor site, and a second guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases downstream of the splice acceptor site.

[0020] Embodiment 13 is the composition of embodiment 12, wherein i. the first guide RNA of the at least one pair of guide RNAs targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases upstream of the splice acceptor site, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases downstream of the splice acceptor site; or ii. the first guide RNA of the at least one pair of guide RNAs targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases upstream of the splice donor site, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases downstream of the splice donor site.

[0021] Embodiment 14 is the composition of embodiment 12 or embodiment 13, wherein a. the fragment of the DMD gene comprises a splice donor site and further comprises a portion of intron 53 and an adjoining portion of exon 53 of the DMD gene; or b. the fragment of the DMD gene comprises a splice acceptor site and further comprises a portion of exon 53 and an adjoining portion of intron 52 of the DMD gene.

[0022] Embodiment 15 is the composition of any one of embodiments 12-14, wherein a. the first guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 1-200 of SEQ ID NO: 144, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 201-400 of SEQ ID NO: 144; or b. the first guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 1-200 of SEQ ID NO: 145, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 201-400 of SEQ ID NO: 145.

[0023] Embodiment 16 is the composition of any one of embodiments 12-15, further comprising a SluCas9 or a nucleic acid encoding a SluCas9.

[0024] Embodiment 17 is a composition comprising one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise i) a guide sequence of any one of the guide sequences shown in Tables 1A-1C; ii) at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of any one of the guide sequences shown in Tables 1A- 1C; iii) a guide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the guide sequences shown in Tables 1A-1C; or iv) a pair of guide RNAs comprising any two of the guide sequence pairs shown in Tables 1E or 1F ; optionally further comprising a SluCas9 or a nucleic acid encoding a SluCas9.

[0025] Embodiment 18 is a composition comprising a first guide RNA and a second guide RNA, wherein a. the first guide RNA is selected from any one of SEQ ID NOs: 1-64; and the second guide RNA is selected from any one of SEQ ID NOs: 65-87; or b. the first guide RNA is selected from any one of SEQ ID NOs: 65-87; and the second guide RNA is selected from any one of SEQ ID NOs: 88-143.

[0026] Embodiment 19 is a composition comprising at least one pair of guide RNAs or a nucleic acid encoding at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprise or consist of a first and a second guide sequence selected from any of the following pairs of first and second guide sequences: a. SEQ ID NOs: 1 and 65; 1 and 66; 1 and 67; 1 and 68; 1 and 69; 1 and 70; 1 and 71; 1 and 72; 1 and 73; 1 and 74; 1 and 75; 1 and 76; 1 and 77; 1 and 78; 1 and 79; 1 and 80; 1 and 81; 1 and 82; 1 and 83; 1 and 84; 1 and 85; 1 and 86; 1 and 87; 2 and 65; 2 and 66; 2 and 67; 2 and 68; 2 and 69; 2 and 70; 2 and 71; 2 and 72; 2 and 73; 2 and 74; 2 and 75; 2 and 76; 2 and 77; 2 and 78; 2 and 79; 2 and 80; 2 and 81; 2 and 82; 2 and 83; 2 and 84; 2 and 85; 2 and 86; 2 and 87; 3 and 65; 3 and 66; 3 and 67; 3 and 68; 3 and 69; 3 and 70; 3 and 71; 3 and 72; 3 and 73; 3 and 74; 3 and 75; 3 and 76; 3 and 77; 3 and 78; 3 and 79; 3 and 80; 3 and 81; 3 and 82; 3 and 83; 3 and 84; 3 and 85; 3 and 86; 3 and 87; 4 and 65; 4 and 66; 4 and 67; 4 and 68; 4 and 69; 4 and 70; 4 and 71; 4 and 72; 4 and 73; 4 and 74; 4 and 75; 4 and 76; 4 and 77; 4 and 78; 4 and 79; 4 and 80; 4 and 81; 4 and 82; 4 and 83; 4 and 84; 4 and 85; 4 and 86; 4 and 87; 5 and 65; 5 and 66; 5 and 67; 5 and 68; 5 and 69; 5 and 70; 5 and 71; 5 and 72; 5 and 73; 5 and 74; 5 and 75; 5 and 76; 5 and 77; 5 and 78; 5 and 79; 5 and 80; 5 and 81; 5 and 82; 5 and 83; 5 and 84; 5 and 85; 5 and 86; 5 and 87; 6 and 65; 6 and 66; 6 and 67; 6 and 68; 6 and 69; 6 and 70; 6 and 71; 6 and 72; 6 and 73; 6 and 74; 6 and 75; 6 and 76; 6 and 77; 6 and 78; 6 and 79; 6 and 80; 6 and 81; 6 and 82; 6 and 83; 6 and 84; 6 and 85; 6 and 86; 6 and 87; 7 and 65; 7 and 66; 7 and 67; 7 and 68; 7 and 69; 7 and 70; 7 and 71; 7 and 72; 7 and 73; 7 and 74; 7 and 75; 7 and 76; 7 and 77; 7 and 78; 7 and 79; 7 and 80; 7 and 81; 7 and 82; 7 and 83; 7 and 84; 7 and 85; 7 and 86; 7 and 87; 8 and 65; 8 and 66; 8 and 67; 8 and 68; 8 and 69; 8 and 70; 8 and 71; 8 and 72; 8 and 73; 8 and 74; 8 and 75; 8 and 76; 8 and 77; 8 and 78; 8 and 79; 8 and 80; 8 and 81; 8 and 82; 8 and 83; 8 and 84; 8 and 85; 8 and 86; 8 and 87; 9 and 65; 9 and 66; 9 and 67; 9 and 68; 9 and 69; 9 and 70; 9 and 71; 9 and 72; 9 and 73; 9 and 74; 9 and 75; 9 and 76; 9 and 77; 9 and 78; 9 and 79; 9 and 80; 9 and 81; 9 and 82; 9 and 83; 9 and 84; 9 and 85; 9 and 86; 9 and 87; 10 and 65; 10 and 66; 10 and 67; 10 and 68; 10 and 69; 10 and 70; 10 and 71; 10 and 72; 10 and 73; 10 and 74; 10 and 75; 10 and 76; 10 and 77; 10 and 78; 10 and 79; 10 and 80; 10 and 81; 10 and 82; 10 and 83; 10 and 84; 10 and 85; 10 and 86; 10 and 87; 11 and 65; 11 and 66; 11 and 67; 11 and 68; 11 and 69; 11 and 70; 11 and 71; 11 and 72; 11 and 73; 11 and 74; 11 and 75; 11 and 76; 11 and 77; 11 and 78; 11 and 79; 11 and 80; 11 and 81; 11 and 82; 11 and 83; 11 and 84; 11 and 85; 11 and 86; 11 and 87; 12 and 65; 12 and 66; 12 and 67; 12 and 68; 12 and 69; 12 and 70; 12 and 71; 12 and 72; 12 and 73; 12 and 74; 12 and 75; 12 and 76; 12 and 77; 12 and 78; 12 and 79; 12 and 80; 12 and 81; 12 and 82; 12 and 83; 12 and 84; 12 and 85; 12 and 86; 12 and 87; 13 and 65; 13 and 66; 13 and 67; 13 and 68; 13 and 69; 13 and 70; 13 and 71; 13 and 72; 13 and 73 ; 13 and 74; 13 and 75; 13 and 76; 13 and 77; 13 and 78; 13 and 79; 13 and 80; 13 and 81; 13 and 82 ; 13 and 83; 13 and 84; 13 and 85; 13 and 86; 13 and 87; 14 and 65; 14 and 66; 14 and 67; 14 and 68 ; 14 and 69; 14 and 70; 14 and 71; 14 and 72; 14 and 73; 14 and 74; 14 and 75; 14 and 76; 14 and 77 ; 14 and 78; 14 and 79; 14 and 80; 14 and 81; 14 and 82; 14 and 83; 14 and 84; 14 and 85; 14 and 86 ; 14 and 87; 15 and 65; 15 and 66; 15 and 67; 15 and 68; 15 and 69; 15 and 70; 15 and 71; 15 and 72 ; 15 and 73; 15 and 74; 15 and 75; 15 and 76; 15 and 77; 15 and 78; 15 and 79; 15 and 80; 15 and 81 ; 15 and 82; 15 and 83; 15 and 84; 15 and 85; 15 and 86; 15 and 87; 16 and 65; 16 and 66; 16 and 67 ; 16 and 68; 16 and 69; 16 and 70; 16 and 71; 16 and 72; 16 and 73; 16 and 74; 16 and 75; 16 and 76 ; 16 and 77; 16 and 78; 16 and 79; 16 and 80; 16 and 81; 16 and 82; 16 and 83; 16 and 84; 16 and 85 ; 16 and 86; 16 and 87; 17 and 65; 17 and 66; 17 and 67; 17 and 68; 17 and 69; 17 and 70; 17 and 71 ; 17 and 72; 17 and 73; 17 and 74; 17 and 75; 17 and 76; 17 and 77; 17 and 78; 17 and 79; 17 and 80 ; 17 and 81; 17 and 82; 17 and 83; 17 and 84; 17 and 85; 17 and 86; 17 and 87; 18 and 65; 18 and 66 ; 18 and 67; 18 and 68; 18 and 69; 18 and 70; 18 and 71 ; 18 and 72; 18 and 73; 18 and 74; 18 and 75 ; 18 and 76; 18 and 77; 18 and 78; 18 and 79; 18 and 80; 18 and 81 ; 18 and 82; 18 and 83; 18 and 84 ; 18 and 85; 18 and 86; 18 and 87; 19 and 65; 19 and 66; 19 and 67; 19 and 68; 19 and 69; 19 and 70 ; 19 and 71; 19 and 72; 19 and 73; 19 and 74; 19 and 75; 19 and 76; 19 and 77; 19 and 78; 19 and 79 ; 19 and 80; 19 and 81; 19 and 82; 19 and 83; 19 and 84; 19 and 85; 19 and 86; 19 and 87; 20 and 65 ; 20 and 66; 20 and 67; 20 and 68; 20 and 69; 20 and 70; 20 and 71 ; 20 and 72; 20 and 73; 20 and 74 ; 20 and 75; 20 and 76; 20 and 77; 20 and 78; 20 and 79; 20 and 80; 20 and 81 ; 20 and 82; 20 and 83 ; 20 and 84; 20 and 85; 20 and 86; 20 and 87; 21 and 65; 21 and 66; 21 and 67; 21 and 68; 21 and 69 ; 21 and 70; 21 and 71; 21 and 72; 21 and 73; 21 and 74; 21 and 75; 21 and 76; 21 and 77; 21 and 78 ; 21 and 79; 21 and 80; 21 and 81; 21 and 82; 21 and 83; 21 and 84; 21 and 85; 21 and 86; 21 and 87 ; 22 and 65; 22 and 66; 22 and 67; 22 and 68; 22 and 69; 22 and 70; 22 and 71 ; 22 and 72; 22 and 73 ; 22 and 74; 22 and 75; 22 and 76; 22 and 77; 22 and 78; 22 and 79; 22 and 80; 22 and 81 ; 22 and 82 ; 22 and 83; 22 and 84; 22 and 85; 22 and 86; 22 and 87; 23 and 65; 23 and 66; 23 and 67 ; 23 and 68 ; 23 and 69; 23 and 70; 23 and 71; 23 and 72; 23 and 73; 23 and 74; 23 and 75; 23 and 76; 23 and 77 ; 23 and 78; 23 and 79; 23 and 80; 23 and 81; 23 and 82; 23 and 83; 23 and 84; 23 and 85; 23 and 86 ; 23 and 87; 24 and 65; 24 and 66; 24 and 67; 24 and 68; 24 and 69; 24 and 70; 24 and 71 ; 24 and 72 ; 24 and 73; 24 and 74; 24 and 75; 24 and 76; 24 and 77; 24 and 78; 24 and 79; 24 and 80; 24 and 81 ; 24 and 82; 24 and 83; 24 and 84; 24 and 85; 24 and 86; 24 and 87; 25 and 65; 25 and 66; 25 and 67 ; 25 and 68; 25 and 69; 25 and 70; 25 and 71; 25 and 72; 25 and 73; 25 and 74; 25 and 75; 25 and 76 ; 25 and 77; 25 and 78; 25 and 79; 25 and 80; 25 and 81; 25 and 82; 25 and 83; 25 and 84; 25 and 85 ; 25 and 86; 25 and 87; 26 and 65; 26 and 66; 26 and 67; 26 and 68; 26 and 69; 26 and 70; 26 and 71 ; 26 and 72; 26 and 73; 26 and 74; 26 and 75; 26 and 76; 26 and 77; 26 and 78; 26 and 79; 26 and 80 ; 26 and 81; 26 and 82; 26 and 83; 26 and 84; 26 and 85; 26 and 86; 26 and 87; 27 and 65; 27 and 66 ; 27 and 67; 27 and 68; 27 and 69; 27 and 70; 27 and 71 ; 27 and 72; 27 and 73; 27 and 74; 27 and 75 ; 27 and 76; 27 and 77; 27 and 78; 27 and 79; 27 and 80; 27 and 81 ; 27 and 82; 27 and 83; 27 and 84 ; 27 and 85; 27 and 86; 27 and 87; 28 and 65; 28 and 66; 28 and 67 ; 28 and 68; 28 and 69; 28 and 70 ; 28 and 71; 28 and 72; 28 and 73; 28 and 74; 28 and 75; 28 and 76; 28 and 77; 28 and 78; 28 and 79 ; 28 and 80; 28 and 81; 28 and 82; 28 and 83; 28 and 84; 28 and 85; 28 and 86; 28 and 87; 29 and 65 ; 29 and 66; 29 and 67; 29 and 68; 29 and 69; 29 and 70; 29 and 71 ; 29 and 72; 29 and 73; 29 and 74 ; 29 and 75; 29 and 76; 29 and 77; 29 and 78; 29 and 79; 29 and 80; 29 and 81 ; 29 and 82; 29 and 83 ; 29 and 84; 29 and 85; 29 and 86; 29 and 87; 30 and 65; 30 and 66; 30 and 67 ; 30 and 68; 30 and 69 ; 30 and 70; 30 and 71; 30 and 72; 30 and 73; 30 and 74; 30 and 75; 30 and 76; 30 and 77; 30 and 78 ; 30 and 79; 30 and 80; 30 and 81; 30 and 82; 30 and 83; 30 and 84; 30 and 85; 30 and 86; 30 and 87 ; 31 and 65; 31 and 66; 31 and 67; 31 and 68; 31 and 69; 31 and 70; 31 and 71 ; 31 and 72; 31 and 73 ; 31 and 74; 31 and 75; 31 and 76; 31 and 77; 31 and 78; 31 and 79; 31 and 80; 31 and 81; 31 and 82 ; 31 and 83; 31 and 84; 31 and 85; 31 and 86; 31 and 87; 32 and 65; 32 and 66; 32 and 67 ; 32 and 68 ; 32 and 69; 32 and 70; 32 and 71; 32 and 72; 32 and 73; 32 and 74; 32 and 75; 32 and 76; 32 and 77 ; 32 and 78; 32 and 79; 32 and 80; 32 and 81; 32 and 82; 32 and 83; 32 and 84; 32 and 85; 32 and 86 ; 32 and 87; 33 and 65; 33 and 66; 33 and 67; 33 and 68; 33 and 69; 33 and 70; 33 and 71; 33 and 72 ; 33 and 73; 33 and 74; 33 and 75; 33 and 76; 33 and 77; 33 and 78; 33 and 79; 33 and 80; 33 and 81 ; 33 and 82; 33 and 83; 33 and 84; 33 and 85; 33 and 86; 33 and 87; 34 and 65; 34 and 66; 34 and 67 ; 34 and 68; 34 and 69; 34 and 70; 34 and 71; 34 and 72; 34 and 73; 34 and 74; 34 and 75; 34 and 76 ; 34 and 77; 34 and 78; 34 and 79; 34 and 80; 34 and 81; 34 and 82; 34 and 83; 34 and 84; 34 and 85 ; 34 and 86; 34 and 87; 35 and 65; 35 and 66; 35 and 67; 35 and 68; 35 and 69; 35 and 70; 35 and 71 ; 35 and 72; 35 and 73; 35 and 74; 35 and 75; 35 and 76; 35 and 77; 35 and 78; 35 and 79; 35 and 80 ; 35 and 81; 35 and 82; 35 and 83; 35 and 84; 35 and 85; 35 and 86; 35 and 87; 36 and 65; 36 and 66 ; 36 and 67; 36 and 68; 36 and 69; 36 and 70; 36 and 71 ; 36 and 72; 36 and 73; 36 and 74; 36 and 75 ; 36 and 76; 36 and 77; 36 and 78; 36 and 79; 36 and 80; 36 and 81; 36 and 82; 36 and 83; 36 and 84 ; 36 and 85; 36 and 86; 36 and 87; 37 and 65; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 37 and 70 ; 37 and 71; 37 and 72; 37 and 73; 37 and 74; 37 and 75; 37 and 76; 37 and 77; 37 and 78; 37 and 79 ; 37 and 80; 37 and 81; 37 and 82; 37 and 83; 37 and 84; 37 and 85; 37 and 86; 37 and 87; 38 and 65 ; 38 and 66; 38 and 67; 38 and 68; 38 and 69; 38 and 70; 38 and 71; 38 and 72; 38 and 73; 38 and 74 ; 38 and 75; 38 and 76; 38 and 77; 38 and 78; 38 and 79; 38 and 80; 38 and 81; 38 and 82; 38 and 83 ; 38 and 84; 38 and 85; 38 and 86; 38 and 87; 39 and 65; 39 and 66; 39 and 67 ; 39 and 68; 39 and 69 ; 39 and 70; 39 and 71; 39 and 72; 39 and 73; 39 and 74; 39 and 75; 39 and 76; 39 and 77; 39 and 78 ; 39 and 79; 39 and 80; 39 and 81; 39 and 82; 39 and 83; 39 and 84; 39 and 85; 39 and 86; 39 and 87 ; 40 and 65; 40 and 66; 40 and 67; 40 and 68; 40 and 69; 40 and 70; 40 and 71 ; 40 and 72; 40 and 73 ; 40 and 74; 40 and 75; 40 and 76; 40 and 77; 40 and 78; 40 and 79; 40 and 80; 40 and 81 ; 40 and 82 ; 40 and 83; 40 and 84; 40 and 85; 40 and 86; 40 and 87; 41 and 65; 41 and 66; 41 and 67; 41 and 68 ; 41 and 69; 41 and 70; 41 and 71; 41 and 72; 41 and 73; 41 and 74; 41 and 75; 41 and 76; 41 and 77 ; 41 and 78; 41 and 79; 41 and 80; 41 and 81; 41 and 82; 41 and 83; 41 and 84; 41 and 85; 41 and 86 ; 41 and 87; 42 and 65; 42 and 66; 42 and 67; 42 and 68; 42 and 69; 42 and 70; 42 and 71 ; 42 and 72 ; 42 and 73; 42 and 74; 42 and 75; 42 and 76; 42 and 77; 42 and 78; 42 and 79; 42 and 80; 42 and 81 ; 42 and 82; 42 and 83; 42 and 84; 42 and 85; 42 and 86; 42 and 87; 43 and 65; 43 and 66; 43 and 67 ; 43 and 68; 43 and 69; 43 and 70; 43 and 71; 43 and 72; 43 and 73; 43 and 74; 43 and 75; 43 and 76 ; 43 and 77; 43 and 78; 43 and 79; 43 and 80; 43 and 81; 43 and 82; 43 and 83; 43 and 84; 43 and 85 ; 43 and 86; 43 and 87; 44 and 65; 44 and 66; 44 and 67; 44 and 68; 44 and 69; 44 and 70; 44 and 71 ; 44 and 72; 44 and 73; 44 and 74; 44 and 75; 44 and 76; 44 and 77; 44 and 78; 44 and 79; 44 and 80 ; 44 and 81; 44 and 82; 44 and 83; 44 and 84; 44 and 85; 44 and 86; 44 and 87; 45 and 65; 45 and 66 ; 45 and 67; 45 and 68; 45 and 69; 45 and 70; 45 and 71 ; 45 and 72; 45 and 73; 45 and 74; 45 and 75 ; 45 and 76; 45 and 77; 45 and 78; 45 and 79; 45 and 80; 45 and 81 ; 45 and 82; 45 and 83; 45 and 84 ; 45 and 85; 45 and 86; 45 and 87; 46 and 65; 46 and 66; 46 and 67 ; 46 and 68; 46 and 69; 46 and 70 ; 46 and 71; 46 and 72; 46 and 73; 46 and 74; 46 and 75; 46 and 76; 46 and 77; 46 and 78; 46 and 79 ; 46 and 80; 46 and 81; 46 and 82; 46 and 83; 46 and 84; 46 and 85; 46 and 86; 46 and 87; 47 and 65 ; 47 and 66; 47 and 67; 47 and 68; 47 and 69; 47 and 70; 47 and 71; 47 and 72; 47 and 73; 47 and 74 ; 47 and 75; 47 and 76; 47 and 77; 47 and 78; 47 and 79; 47 and 80; 47 and 81 ; 47 and 82; 47 and 83 ; 47 and 84; 47 and 85; 47 and 86; 47 and 87; 48 and 65; 48 and 66; 48 and 67 ; 48 and 68; 48 and 69 ; 48 and 70; 48 and 71; 48 and 72; 48 and 73; 48 and 74; 48 and 75; 48 and 76; 48 and 77; 48 and 78 ; 48 and 79; 48 and 80; 48 and 81; 48 and 82; 48 and 83; 48 and 84; 48 and 85; 48 and 86; 48 and 87 ; 49 and 65; 49 and 66; 49 and 67; 49 and 68; 49 and 69; 49 and 70; 49 and 71 ; 49 and 72; 49 and 73 ; 49 and 74; 49 and 75; 49 and 76; 49 and 77; 49 and 78; 49 and 79; 49 and 80; 49 and 81 ; 49 and 82 ; 49 and 83; 49 and 84; 49 and 85; 49 and 86; 49 and 87; 50 and 65; 50 and 66; 50 and 67 ; 50 and 68 ; 50 and 69; 50 and 70; 50 and 71; 50 and 72; 50 and 73; 50 and 74; 50 and 75; 50 and 76; 50 and 77 ; 50 and 78; 50 and 79; 50 and 80; 50 and 81; 50 and 82; 50 and 83; 50 and 84; 50 and 85; 50 and 86 ; 50 and 87; 51 and 65; 51 and 66; 51 and 67; 51 and 68; 51 and 69; 51 and 70; 51 and 71; 51 and 72 ; 51 and 73; 51 and 74; 51 and 75; 51 and 76; 51 and 77; 51 and 78; 51 and 79; 51 and 80; 51 and 81 ; 51 and 82; 51 and 83; 51 and 84; 51 and 85; 51 and 86; 51 and 87; 52 and 65; 52 and 66; 52 and 67 ; 52 and 68; 52 and 69; 52 and 70; 52 and 71; 52 and 72; 52 and 73; 52 and 74; 52 and 75; 52 and 76 ; 52 and 77; 52 and 78; 52 and 79; 52 and 80; 52 and 81; 52 and 82; 52 and 83; 52 and 84; 52 and 85 ; 52 and 86; 52 and 87; 53 and 65; 53 and 66; 53 and 67; 53 and 68; 53 and 69; 53 and 70; 53 and 71 ; 53 and 72; 53 and 73; 53 and 74; 53 and 75; 53 and 76; 53 and 77; 53 and 78; 53 and 79; 53 and 80 ; 53 and 81; 53 and 82; 53 and 83; 53 and 84; 53 and 85; 53 and 86; 53 and 87; 54 and 65; 54 and 66 ; 54 and 67; 54 and 68; 54 and 69; 54 and 70; 54 and 71 ; 54 and 72; 54 and 73; 54 and 74; 54 and 75 ; 54 and 76; 54 and 77; 54 and 78; 54 and 79; 54 and 80; 54 and 81 ; 54 and 82; 54 and 83; 54 and 84 ; 54 and 85; 54 and 86; 54 and 87; 55 and 65; 55 and 66; 55 and 67 ; 55 and 68; 55 and 69; 55 and 70 ; 55 and 71; 55 and 72; 55 and 73; 55 and 74; 55 and 75; 55 and 76; 55 and 77; 55 and 78; 55 and 79 ; 55 and 80; 55 and 81; 55 and 82; 55 and 83; 55 and 84; 55 and 85; 55 and 86; 55 and 87; 56 and 65 ; 56 and 66; 56 and 67; 56 and 68; 56 and 69; 56 and 70; 56 and 71; 56 and 72; 56 and 73; 56 and 74 ; 56 and 75; 56 and 76; 56 and 77; 56 and 78; 56 and 79; 56 and 80; 56 and 81; 56 and 82; 56 and 83; 56 and 84; 56 and 85; 56 and 86; 56 and 87; 57 and 65; 57 and 66; 57 and 67; 57 and 68; 57 and 69; 57 and 70; 57 and 71; 57 and 72; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 57 and 78; 57 and 79; 57 and 80; 57 and 81; 57 and 82; 57 and 83; 57 and 84; 57 and 85; 57 and 86; 57 and 87; 58 and 65; 58 and 66; 58 and 67; 58 and 68; 58 and 69; 58 and 70; 58 and 71; 58 and 72; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 58 and 78; 58 and 79; 58 and 80; 58 and 81; 58 and 82; 58 and 83; 58 and 84; 58 and 85; 58 and 86; 58 and 87; 59 and 65; 59 and 66; 59 and 67; 59 and 68; 59 and 69; 59 and 70; 59 and 71; 59 and 72; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 59 and 78; 59 and 79; 59 and 80; 59 and 81; 59 and 82; 59 and 83; 59 and 84; 59 and 85; 59 and 86; 59 and 87; 60 and 65; 60 and 66; 60 and 67; 60 and 68; 60 and 69; 60 and 70; 60 and 71; 60 and 72; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 60 and 78; 60 and 79; 60 and 80; 60 and 81; 60 and 82; 60 and 83; 60 and 84; 60 and 85; 60 and 86; 60 and 87; 61 and 65; 61 and 66; 61 and 67; 61 and 68; 61 and 69; 61 and 70; 61 and 71; 61 and 72; 61 and 73; 61 and 74; 61 and 75; 61 and 76; 61 and 77; 61 and 78; 61 and 79; 61 and 80; 61 and 81; 61 and 82; 61 and 83; 61 and 84; 61 and 85; 61 and 86; 61 and 87; 62 and 65; 62 and 66; 62 and 67; 62 and 68; 62 and 69; 62 and 70; 62 and 71; 62 and 72; 62 and 73; 62 and 74; 62 and 75; 62 and 76; 62 and 77; 62 and 78; 62 and 79; 62 and 80; 62 and 81; 62 and 82; 62 and 83; 62 and 84; 62 and 85; 62 and 86; 62 and 87; 63 and 65; 63 and 66; 63 and 67; 63 and 68; 63 and 69; 63 and 70; 63 and 71; 63 and 72; 63 and 73; 63 and 74; 63 and 75; 63 and 76; 63 and 77; 63 and 78; 63 and 79; 63 and 80; 63 and 81; 63 and 82; 63 and 83; 63 and 84; 63 and 85; 63 and 86; 63 and 87; 64 and 65; 64 and 66; 64 and 67; 64 and 68; 64 and 69; 64 and 70; 64 and 71; 64 and 72; 64 and 73; 64 and 74; 64 and 75; 64 and 76; 64 and 77; 64 and 78; 64 and 79; 64 and 80; 64 and 81; 64 and 82; 64 and 83; 64 and 84; 64 and 85; 64 and 86; or 64 and 87; b. SEQ ID NOs: 88 and 65; 88 and 66; 88 and 67; 88 and 68; 88 and 69; 88 and 70; 88 and 71; 88 and 72; 88 and 73; 88 and 74; 88 and 75; 88 and 76; 88 and 77; 88 and 78; 88 and 79; 88 and 80;

88 and 81; 88 and 82; 88 and 83; 88 and 84; 88 and 85; 88 and 86; 88 and 87; 89 and 65; 89 and 66;

89 and 67; 89 and 68; 89 and 69; 89 and 70; 89 and 71; 89 and 72; 89 and 73; 89 and 74; 89 and 75;

89 and 76; 89 and 77; 89 and 78; 89 and 79; 89 and 80; 89 and 81; 89 and 82; 89 and 83; 89 and 84;

89 and 85; 89 and 86; 89 and 87; 90 and 65; 90 and 66; 90 and 67; 90 and 68; 90 and 69; 90 and 70;

90 and 71; 90 and 72; 90 and 73; 90 and 74; 90 and 75; 90 and 76; 90 and 77; 90 and 78; 90 and 79;

90 and 80; 90 and 81; 90 and 82; 90 and 83; 90 and 84; 90 and 85; 90 and 86; 90 and 87; 91 and 65;

91 and 66; 91 and 67; 91 and 68; 91 and 69; 91 and 70; 91 and 71; 91 and 72; 91 and 73; 91 and 74;

91 and 75; 91 and 76; 91 and 77; 91 and 78; 91 and 79; 91 and 80; 91 and 81; 91 and 82; 91 and 83;

91 and 84; 91 and 85; 91 and 86; 91 and 87; 92 and 65; 92 and 66; 92 and 67; 92 and 68; 92 and 69;

92 and 70; 92 and 71; 92 and 72; 92 and 73; 92 and 74; 92 and 75; 92 and 76; 92 and 77; 92 and 78;

92 and 79; 92 and 80; 92 and 81; 92 and 82; 92 and 83; 92 and 84; 92 and 85; 92 and 86; 92 and 87;

93 and 65; 93 and 66; 93 and 67; 93 and 68; 93 and 69; 93 and 70; 93 and 71; 93 and 72; 93 and 73;

93 and 74; 93 and 75; 93 and 76; 93 and 77; 93 and 78; 93 and 79; 93 and 80; 93 and 81; 93 and 82; and 83; 93 and 84; 93 and 85; 93 and 86; 93 and 87; 94 and 65; 94 and 66; 94 and 67 ; 94 and 68 and 69; 94 and 70; 94 and 71; 94 and 72; 94 and 73; 94 and 74; 94 and 75; 94 and 76; 94 and 77 and 78; 94 and 79; 94 and 80; 94 and 81; 94 and 82; 94 and 83; 94 and 84; 94 and 85; 94 and 86 and 87; 95 and 65; 95 and 66; 95 and 67; 95 and 68; 95 and 69; 95 and 70; 95 and 71; 95 and 72 and 73; 95 and 74; 95 and 75; 95 and 76; 95 and 77; 95 and 78; 95 and 79; 95 and 80; 95 and 81 and 82; 95 and 83; 95 and 84; 95 and 85; 95 and 86; 95 and 87; 96 and 65; 96 and 66; 96 and 67 and 68; 96 and 69; 96 and 70; 96 and 71; 96 and 72; 96 and 73; 96 and 74; 96 and 75; 96 and 76 and 77; 96 and 78; 96 and 79; 96 and 80; 96 and 81; 96 and 82; 96 and 83; 96 and 84; 96 and 85 and 86; 96 and 87; 97 and 65; 97 and 66; 97 and 67; 97 and 68; 97 and 69; 97 and 70; 97 and 71 and 72; 97 and 73; 97 and 74; 97 and 75; 97 and 76; 97 and 77; 97 and 78; 97 and 79; 97 and 80 and 81; 97 and 82; 97 and 83; 97 and 84; 97 and 85; 97 and 86; 97 and 87; 98 and 65; 98 and 66 and 67; 98 and 68; 98 and 69; 98 and 70; 98 and 71; 98 and 72; 98 and 73; 98 and 74; 98 and 75 and 76; 98 and 77; 98 and 78; 98 and 79; 98 and 80; 98 and 81; 98 and 82; 98 and 83; 98 and 84 and 85; 98 and 86; 98 and 87; 99 and 65; 99 and 66; 99 and 67; 99 and 68; 99 and 69; 99 and 70 and 71; 99 and 72; 99 and 73; 99 and 74; 99 and 75; 99 and 76; 99 and 77; 99 and 78; 99 and 79 and 80; 99 and 81; 99 and 82; 99 and 83; 99 and 84; 99 and 85; ' 19 and 86; 99 and 87; 100 and 65 and 66; 100 and 67; 100 and 68; 100 and 69; 100 and 70; 100 and 71; 100 and 72; 100 and 73; and 74; 100 and 75; 100 and 76; 100 and 77; 100 and 78; 100 and 79; 100 and 80; 100 and 81; and 82; 100 and 83; 100 and 84; 100 and 85; 100 and 86; 100 and 87; 101 and 65; 101 and 66; and 67; 101 and 68; 101 and 69; 101 and 70; 101 and 71; 101 and 72; 101 and 73; 101 and 74; and 75; 101 and 76; 101 and 77; 101 and 78; 101 and 79; 101 and 80; 101 and 81; 101 and 82; and 83; 101 and 84; 101 and 85; 101 and 86; 101 and 87; 102 and 65; 102 and 66; 102 and 67; and 68; 102 and 69; 102 and 70; 102 and 71; 102 and 72; 102 and 73; 102 and 74; 102 and 75; and 76; 102 and 77; 102 and 78; 102 and 79; 102 and 80; 102 and 81; 102 and 82; 102 and 83; and 84; 102 and 85; 102 and 86; 102 and 87; 103 and 65; 103 and 66; 103 and 67; 103 and 68; and 69; 103 and 70; 103 and 71; 103 and 72; 103 and 73; 103 and 74; 103 and 75; 103 and 76; and 77; 103 and 78; 103 and 79; 103 and 80; 103 and 81; 103 and 82; 103 and 83; 103 and 84; and 85; 103 and 86; 103 and 87; 104 and 65; 104 and 66; 104 and 67; 104 and 68; 104 and 69; and 70; 104 and 71; 104 and 72; 104 and 73; 104 and 74; 104 and 75; 104 and 76; 104 and 77; and 78; 104 and 79; 104 and 80; 104 and 81; 104 and 82; 104 and 83; 104 and 84; 104 and 85; and 86; 104 and 87; 105 and 65; 105 and 66; 105 and 67; 105 and 68; 105 and 69; 105 and 70; and 71; 105 and 72; 105 and 73; 105 and 74; 105 and 75; 105 and 76; 105 and 77; 105 and 78; and 79; 105 and 80; 105 and 81; 105 and 82; 105 and 83; 105 and 84; 105 and 85; 105 and 86; and 87; 106 and 65; 106 and 66; 106 and 67; 106 and 68; 106 and 69; 106 and 70; 106 and 71; and 72; 106 and 73; 106 and 74; 106 and 75; 106 and 76; 106 and 77; 106 and 78; 106 and 79; and 80; 106 and 81; 106 and 82; 106 and 83; 106 and 84; 106 and 85; 106 and 86; 106 and 87; and 65; 107 and 66; 107 and 67; 107 and 68; 107 and 69; 107 and 70; 107 and 71; 107 and 72; and 73; 107 and 74; 107 and 75; 107 and 76; 107 and 77; 107 and 78; 107 and 79; 107 and 80; and 81; 107 and 82; 107 and 83; 107 and 84; 107 and 85; 107 and 86; 107 and 87; 108 and 65; and 66; 108 and 67; 108 and 68; 108 and 69; 108 and 70; 108 and 71; 108 and 72; 108 and 73; and 74; 108 and 75; 108 and 76; 108 and 77; 108 and 78; 108 and 79; 108 and 80; 108 and 81; and 82; 108 and 83; 108 and 84; 108 and 85; 108 and 86; 108 and 87; 109 and 65; 109 and 66; and 67; 109 and 68; 109 and 69; 109 and 70; 109 and 71; 109 and 72; 109 and 73; 109 and 74; and 75; 109 and 76; 109 and 77; 109 and 78; 109 and 79; 109 and 80; 109 and 81; 109 and 82; and 83; 109 and 84; 109 and 85; 109 and 86; 109 and 87; 110 and 65; 110 and 66; 110 and 67; and 68; 110 and 69; 110 and 70; 110 and 71; 110 and 72; 110 and 73; 110 and 74; 110 and 75; and 76; 110 and 77; 110 and 78; 110 and 79; 110 and 80; 110 and 81; 110 and 82; 110 and 83; and 84; 110 and 85; 110 and 86; 110 and 87; 111 and 65; 111 and 66; 111 and 67; 111 and 68; and 69; 111 and 70; 111 and 71; 111 and 72; 111 and 73; 111 and 74; 111 and 75; 111 and 76; and 77; 111 and 78; 111 and 79; 111 and 80; 111 and 81; 111 and 82; 111 and 83; 111 and 84; and 85; 111 and 86; 111 and 87; 112 and 65; 112 and 66; 112 and 67; 112 and 68; 112 and 69; and 70; 112 and 71; 112 and 72; 112 and 73; 112 and 74; 112 and 75; 112 and 76; 112 and 77; and 78; 112 and 79; 112 and 80; 112 and 81; 112 and 82; 112 and 83; 112 and 84; 112 and 85; and 86; 112 and 87; 113 and 65; 113 and 66; 113 and 67; 113 and 68; 113 and 69; 113 and 70; and 71; 113 and 72; 113 and 73; 113 and 74; 113 and 75; 113 and 76; 113 and 77; 113 and 78; and 79; 113 and 80; 113 and 81; 113 and 82; 113 and 83; 113 and 84; 113 and 85; 113 and 86; and 87; 114 and 65; 114 and 66; 114 and 67; 114 and 68; 114 and 69; 114 and 70; 114 and 71; and 72; 114 and 73; 114 and 74; 114 and 75; 114 and 76; 114 and 77; 114 and 78; 114 and 79; and 80; 114 and 81; 114 and 82; 114 and 83; 114 and 84; 114 and 85; 114 and 86; 114 and 87; and 65; 115 and 66; 115 and 67; 115 and 68; 115 and 69; 115 and 70; 115 and 71; 115 and 72; and 73; 115 and 74; 115 and 75; 115 and 76; 115 and 77; 115 and 78; 115 and 79; 115 and 80; and 81; 115 and 82; 115 and 83; 115 and 84; 115 and 85; 115 and 86; 115 and 87; 116 and 65; and 66; 116 and 67; 116 and 68; 116 and 69; 116 and 70; 116 and 71; 116 and 72; 116 and 73; and 74; 116 and 75; 116 and 76; 116 and 77; 116 and 78; 116 and 79; 116 and 80; 116 and 81; and 82; 116 and 83; 116 and 84; 116 and 85; 116 and 86; 116 and 87; 117 and 65; 117 and 66; and 67; 117 and 68; 117 and 69; 117 and 70; 117 and 71; 117 and 72; 117 and 73; 117 and 74; and 75; 117 and 76; 117 and 77; 117 and 78; 117 and 79; 117 and 80; 117 and 81; 117 and 82; and 83; 117 and 84; 117 and 85; 117 and 86; 117 and 87; 118 and 65; 118 and 66; 118 and 67; and 68; 118 and 69; 118 and 70; 118 and 71; 118 and 72; 118 and 73; 118 and 74; 118 and 75; and 76; 118 and 77; 118 and 78; 118 and 79; 118 and 80; 118 and 81; 118 and 82; 118 and 83; and 84; 118 and 85; 118 and 86; 118 and 87; 119 and 65; 119 and 66; 119 and 67; 119 and 68; and 69; 119 and 70; 119 and 71; 119 and 72; 119 and 73; 119 and 74; 119 and 75; 119 and 76; and 77; 119 and 78; 119 and 79; 119 and 80; 119 and 81; 119 and 82; 119 and 83; 119 and 84; and 85; 119 and 86; 119 and 87; 120 and 65; 120 and 66; 120 and 67; 120 and 68; 120 and 69; and 70; 120 and 71; 120 and 72; 120 and 73; 120 and 74; 120 and 75; 120 and 76; 120 and 77; and 78; 120 and 79; 120 and 80; 120 and 81; 120 and 82; 120 and 83; 120 and 84; 120 and 85; and 86; 120 and 87; 121 and 65; 121 and 66; 121 and 67; 121 and 68; 121 and 69; 121 and 70; and 71; 121 and 72; 121 and 73; 121 and 74; 121 and 75; 121 and 76; 121 and 77; 121 and 78; and 79; 121 and 80; 121 and 81; 121 and 82; 121 and 83; 121 and 84; 121 and 85; 121 and 86; and 87; 122 and 65; 122 and 66; 122 and 67; 122 and 68; 122 and 69; 122 and 70; 122 and 71; and 72; 122 and 73; 122 and 74; 122 and 75; 122 and 76; 122 and 77; 122 and 78; 122 and 79; and 80; 122 and 81; 122 and 82; 122 and 83; 122 and 84; 122 and 85; 122 and 86; 122 and 87; and 65; 123 and 66; 123 and 67; 123 and 68; 123 and 69; 123 and 70; 123 and 71; 123 and 72; and 73; 123 and 74; 123 and 75; 123 and 76; 123 and 77; 123 and 78; 123 and 79; 123 and 80; and 81; 123 and 82; 123 and 83; 123 and 84; 123 and 85; 123 and 86; 123 and 87; 124 and 65; and 66; 124 and 67; 124 and 68; 124 and 69; 124 and 70; 124 and 71; 124 and 72; 124 and 73; and 74; 124 and 75; 124 and 76; 124 and 77; 124 and 78; 124 and 79; 124 and 80; 124 and 81; and 82; 124 and 83; 124 and 84; 124 and 85; 124 and 86; 124 and 87; 125 and 65; 125 and 66; and 67; 125 and 68; 125 and 69; 125 and 70; 125 and 71; 125 and 72; 125 and 73; 125 and 74; and 75; 125 and 76; 125 and 77; 125 and 78; 125 and 79; 125 and 80; 125 and 81; 125 and 82; and 83; 125 and 84; 125 and 85; 125 and 86; 125 and 87; 126 and 65; 126 and 66; 126 and 67; and 68; 126 and 69; 126 and 70; 126 and 71; 126 and 72; 126 and 73; 126 and 74; 126 and 75; and 76; 126 and 77; 126 and 78; 126 and 79; 126 and 80; 126 and 81; 126 and 82; 126 and 83; and 84; 126 and 85; 126 and 86; 126 and 87; 127 and 65; 127 and 66; 127 and 67; 127 and 68; and 69; 127 and 70; 127 and 71; 127 and 72; 127 and 73; 127 and 74; 127 and 75; 127 and 76; and 77; 127 and 78; 127 and 79; 127 and 80; 127 and 81; 127 and 82; 127 and 83; 127 and 84; and 85; 127 and 86; 127 and 87; 128 and 65; 128 and 66; 128 and 67; 128 and 68; 128 and 69; and 70; 128 and 71; 128 and 72; 128 and 73; 128 and 74; 128 and 75; 128 and 76; 128 and 77; and 78; 128 and 79; 128 and 80; 128 and 81; 128 and 82; 128 and 83; 128 and 84; 128 and 85; and 86; 128 and 87; 129 and 65; 129 and 66; 129 and 67; 129 and 68; 129 and 69; 129 and 70; and 71; 129 and 72; 129 and 73; 129 and 74; 129 and 75; 129 and 76; 129 and 77; 129 and 78; and 79; 129 and 80; 129 and 81; 129 and 82; 129 and 83; 129 and 84; 129 and 85; 129 and 86; and 87; 130 and 65; 130 and 66; 130 and 67; 130 and 68; 130 and 69; 130 and 70; 130 and 71; and 72; 130 and 73; 130 and 74; 130 and 75; 130 and 76; 130 and 77; 130 and 78; 130 and 79; and 80; 130 and 81; 130 and 82; 130 and 83; 130 and 84; 130 and 85; 130 and 86; 130 and 87; and 65; 131 and 66; 131 and 67; 131 and 68; 131 and 69; 131 and 70; 131 and 71; 131 and 72; and 73; 131 and 74; 131 and 75; 131 and 76; 131 and 77; 131 and 78; 131 and 79; 131 and 80; and 81; 131 and 82; 131 and 83; 131 and 84; 131 and 85; 131 and 86; 131 and 87; 132 and 65; and 66; 132 and 67; 132 and 68; 132 and 69; 132 and 70; 132 and 71; 132 and 72; 132 and 73; and 74; 132 and 75; 132 and 76; 132 and 77; 132 and 78; 132 and 79; 132 and 80; 132 and 81; and 82; 132 and 83; 132 and 84; 132 and 85; 132 and 86; 132 and 87; 133 and 65; 133 and 66; 133 and 67; 133 and 68; 133 and 69; 133 and 70; 133 and 71; 133 and 72; 133 and 73; 133 and 74;

133 and 75; 133 and 76; 133 and 77; 133 and 78; 133 and 79; 133 and 80; 133 and 81; 133 and 82;

133 and 83; 133 and 84; 133 and 85; 133 and 86; 133 and 87; 134 and 65; 134 and 66; 134 and 67;

134 and 68; 134 and 69; 134 and 70; 134 and 71; 134 and 72; 134 and 73; 134 and 74; 134 and 75;

134 and 76; 134 and 77; 134 and 78; 134 and 79; 134 and 80; 134 and 81; 134 and 82; 134 and 83;

134 and 84; 134 and 85; 134 and 86; 134 and 87; 135 and 65; 135 and 66; 135 and 67; 135 and 68;

135 and 69; 135 and 70; 135 and 71; 135 and 72; 135 and 73; 135 and 74; 135 and 75; 135 and 76;

135 and 77; 135 and 78; 135 and 79; 135 and 80; 135 and 81; 135 and 82; 135 and 83; 135 and 84;

135 and 85; 135 and 86; 135 and 87; 136 and 65; 136 and 66; 136 and 67; 136 and 68; 136 and 69;

136 and 70; 136 and 71; 136 and 72; 136 and 73; 136 and 74; 136 and 75; 136 and 76; 136 and 77;

136 and 78; 136 and 79; 136 and 80; 136 and 81; 136 and 82; 136 and 83; 136 and 84; 136 and 85;

136 and 86; 136 and 87; 137 and 65; 137 and 66; 137 and 67; 137 and 68; 137 and 69; 137 and 70;

137 and 71; 137 and 72; 137 and 73; 137 and 74; 137 and 75; 137 and 76; 137 and 77; 137 and 78;

137 and 79; 137 and 80; 137 and 81; 137 and 82; 137 and 83; 137 and 84; 137 and 85; 137 and 86;

137 and 87; 138 and 65; 138 and 66; 138 and 67; 138 and 68; 138 and 69; 138 and 70; 138 and 71;

138 and 72; 138 and 73; 138 and 74; 138 and 75; 138 and 76; 138 and 77; 138 and 78; 138 and 79;

138 and 80; 138 and 81; 138 and 82; 138 and 83; 138 and 84; 138 and 85; 138 and 86; 138 and 87;

139 and 65; 139 and 66; 139 and 67; 139 and 68; 139 and 69; 139 and 70; 139 and 71; 139 and 72;

139 and 73; 139 and 74; 139 and 75; 139 and 76; 139 and 77; 139 and 78; 139 and 79; 139 and 80;

139 and 81; 139 and 82; 139 and 83; 139 and 84; 139 and 85; 139 and 86; 139 and 87; 140 and 65;

140 and 66; 140 and 67; 140 and 68; 140 and 69; 140 and 70; 140 and 71; 140 and 72; 140 and 73;

140 and 74; 140 and 75; 140 and 76; 140 and 77; 140 and 78; 140 and 79; 140 and 80; 140 and 81;

140 and 82; 140 and 83; 140 and 84; 140 and 85; 140 and 86; 140 and 87; 141 and 65; 141 and 66;

141 and 67; 141 and 68; 141 and 69; 141 and 70; 141 and 71; 141 and 72; 141 and 73; 141 and 74;

141 and 75; 141 and 76; 141 and 77; 141 and 78; 141 and 79; 141 and 80; 141 and 81; 141 and 82;

141 and 83; 141 and 84; 141 and 85; 141 and 86; 141 and 87; 142 and 65; 142 and 66; 142 and 67;

142 and 68; 142 and 69; 142 and 70; 142 and 71; 142 and 72; 142 and 73; 142 and 74; 142 and 75;

142 and 76; 142 and 77; 142 and 78; 142 and 79; 142 and 80; 142 and 81; 142 and 82; 142 and 83;

142 and 84; 142 and 85; 142 and 86; 142 and 87; 143 and 65; 143 and 66; 143 and 67; 143 and 68;

143 and 69; 143 and 70; 143 and 71; 143 and 72; 143 and 73; 143 and 74; 143 and 75; 143 and 76;

143 and 77; 143 and 78; 143 and 79; 143 and 80; 143 and 81; 143 and 82; 143 and 83; 143 and 84;

143 and 85; 143 and 86; or 143 and 87; c. SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and 80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70; 57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76; 58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; or d. SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82; 121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and 83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and 84; 119 and 84; 120 and 84; 121 and 84; 90 and 84; 115 and 85; 116 and 85; 117 and 85; 118 and 85; 119 and 85; 120 and 85; 121 and 85; 90 and 85; 115 and 68; 116 and 68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and 68; 90 and 68; 115 and 69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and 70; 90 and 70;

115 and 71; 116 and 71; 117 and 71; 118 and 71; 119 and 71; 120 and 71; 121 and 71; 90 and 71; 115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and 75;

116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76; 116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and 81;

117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81.

[0027] Embodiment 20 is a composition comprising one or more nucleic acid molecules encoding a. a Staphylococcus lugdunensis Cas9 (SluCas9); and b. at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first guide sequence and a second guide sequence selected from any of the pairs of guide sequences shown in Tables IE or 1F.

[0028] Embodiment 21 is a composition comprising: a. a single nucleic acid molecule comprising: i. a nucleic acid encoding Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; or ii. a nucleic acid encoding SluCas9 and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii. a nucleic acid encoding SluCas9 and 1, 2, or 3 guide RNAs; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A-1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide sequence selected from the pairs of guide sequences shown in Tables 1E or 1F, and further optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence; or b. two nucleic acid molecules comprising: i. a first nucleic acid encoding SluCas9; and a second nucleic acid that does not encode a SluCas9 and encodes any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; ii. a first nucleic acid encoding SluCas9 and

1. at least one, at least two, or at least three guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. 1, 2, or 3 guide RNAs; and a second nucleic acid that does not encode a SluCas9, optionally wherein the second nucleic acid comprises any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; iii. a first nucleic acid encoding SluCas9 and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SluCas9 and encodes from one to six guide RNAs; or iv. a first nucleic acid encoding SluCas9 and at least two guide RNAs, and a second nucleic acid that does not encode a SluCas9 and encodes at least one additional copy of each of the guide RNAs encoded in the first nucleic acid; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A-1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a guide sequence selected from the pairs of guide sequences shown in Tables 1E or 1F, and optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence.

[0029] Embodiment 22 is a composition comprising a single nucleic acid molecule encoding at least one pair of guide RNAs and a Cas9, wherein the single nucleic acid molecule comprises: a. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences shown in Table 1E or 1 F; and a second nucleic acid encoding a Staphylococcus lugdunensis Cas9 (SluCas9); b. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences of: SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and 80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70;

38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70;

36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82;

35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80;

39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76;

35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69;

37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75;

38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71;

19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71;

20 and 84; 20 and 70; 57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83;

57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80;

57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76; 58 and 76; 19 and 76;

57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73;

19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; and a second nucleic acid encoding a SluCas9; or c. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences of: SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82; 121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and 83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and 84; 119 and 84; 120 and 84; 121 and 84; 90 and 84; 115 and 85; 116 and 85; 117 and 85; 118 and 85; 119 and 85; 120 and 85; 121 and 85; 90 and 85; 115 and 68; 116 and 68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and 68; 90 and 68; 115 and 69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and 70; 90 and

70; 115 and 71; 116 and 71; 117 and 71; 118 and 71; 119 and 71; 120 and 71; 121 and 71; 90 and 71;

115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and

75; 116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76;

116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and

81; 117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81; and a second nucleic acid encoding a SluCas9.

[0030] Embodiment 23 is a composition comprising a first and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a Staphylococcus lugdunensis Cas9 (SluCas9) and optionally a first or a first and second guide RNA, and the second nucleic acid molecule comprises a first or a first and second guide RNA, wherein the first guide RNA comprises a first guide sequence and the second guide RNA comprises a second guide sequence selected from any one the following pairs of first and second guide sequences: a. any one of the pairs of first and second guide sequences shown in Tables IE or 1F; b. SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and

80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70; 57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76; 58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; or c. SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82;

121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and 83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and 84; 119 and 84; 120 and 84; 121 and 84; 90 and 84; 115 and 85; 116 and 85; 117 and 85; 118 and 85; 119 and 85; 120 and 85; 121 and 85; 90 and 85; 115 and 68; 116 and 68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and 68; 90 and 68; 115 and 69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and 70; 90 and 70;

115 and 71; 116 and 71; 117 and 71; 118 and 71; 119 and 71; 120 and 71; 121 and 71; 90 and 71; 115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and 75;

116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76; 116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and 81;

117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81.

[0031] Embodiment 24 is a composition comprising one or more nucleic acid molecules encoding a Staphylococcus lugdunensis Cas9 (SluCas9) and at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide RNA, wherein the first guide RNA targets a sequence in intron 53 of a DMD gene and the second guide RNA targets a sequence in exon 53 of the DMD gene, or the first guide RNA targets a sequence in exon 53 of a DMD gene and the second guide RNA targets a sequence in intron 52 of the DMD gene.

[0032] Embodiment 25 is the composition of any one of embodiments 12-24, comprising at least one pair of guide RNAs, wherein the at least one pair of guide RNAs is capable of excising a fragment of the DMD gene, wherein the fragment comprises a splice donor site or a splice acceptor site, and optionally wherein the fragment is between 5 and 400 nucleotides in length.

[0033] Embodiment 26 is the composition of any one of embodiments 12-15 or 25, wherein the excised fragment of the DMD gene does not comprise an entire exon of the DMD gene.

[0034] Embodiment 27 is the composition of any one of embodiments 12-26, comprising at least one pair of guide RNAs, wherein the first and the second guide RNAs of the at least one pair of guide RNAs each comprise a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a first and a second guide sequence selected from any one of the following pairs of first and second guide sequences: a. any one of the pairs of first and second guide sequences shown in Tables IE or 1F; b. SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and

80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70; 57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76; 58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; or c. SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82; 121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and 83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and 84; 119 and 84; 120 and 84; 121 and 84; 90 and 84; 115 and 85; 116 and 85; 117 and 85; 118 and 85; 119 and 85; 120 and 85; 121 and 85; 90 and 85; 115 and 68; 116 and 68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and 68; 90 and 68; 115 and 69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and 70; 90 and 70;

115 and 71; 116 and 71; 117 and 71; 118 and 71; 119 and 71; 120 and 71; 121 and 71; 90 and 71; 115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and 75;

116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76; 116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and 81;

117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81.

[0035] Embodiment 28 is the composition of any one of embodiments 12-26, wherein: i) the first guide RNA comprises the sequence of SEQ ID NO: 70 and the second guide RNA comprises the sequence of SEQ ID NO: 2; ii) the first guide RNA comprises the sequence of SEQ ID NO: 76, and the second guide RNA comprises the sequence of SEQ ID NO: 2; or iii) the first guide RNA comprises the sequence of SEQ ID NO: 115, and the second guide RNA comprises the sequence of SEQ ID NO: 70.

[0036] Embodiment 29 is the composition of any one of the preceding embodiments, comprising at least one pair of guide RNAs, wherein once expressed in vitro or in vivo in combination with a SluCas9 endonuclease, the at least one pair of guide RNAs (i) excises exon 53 of a DMD gene, (ii) excises a fragment of the DMD gene comprising a portion of intron 53 and an adjoining portion of exon 53, wherein the excised fragment of the DMD gene comprises a splice donor site, or (iii) excises a fragment of the DMD gene comprising a portion of exon 53 and an adjoining portion of intron 52, wherein the excised fragment of the DMD gene comprises a splice acceptor site; wherein the size of the excised fragment of the DMD gene is between 5 and 400 nucleotides in length.

[0037] Embodiment 30 is the composition of any one of embodiments 12-15 or 25-29, wherein the size of the excised fragment of the DMD gene is between 5 and 400, 5 and 375, 5 and 350, 5 and 325, 5 and 300, 5 and 275, 5 and 250, 5 and 225, 5 and 200, 5 and 175, 5 and 150, 5 and 125, 5 and

100, 20 and 400, 20 and 375, 20 and 350, 20 and 325, 20 and 300, 20 and 275, 20 and 250, 20 and

225, 20 and 200, 20 and 175, 20 and 150, 20 and 125, 20 and 100, 50 and 400, 50 and 375, 50 and

350, 50 and 325, 50 and 300, 50 and 275, 50 and 250, 50 and 225, 50 and 200, 50 and 175, 50 and

150, 50 and 125, or 50 and 100 nucleotides in length.

[0038] Embodiment 31 is the composition of any one of the preceding embodiments, wherein the one or more guide RNAs is an sgRNA, the first and/or second guide RNA is an sgRNA, or the at least one pair of guide RNAs is a pair of sgRNAs.

[0039] Embodiment 32 is the composition of any one of the preceding embodiments, wherein the one or more guide RNAs is modified, the first and/or second guide RNA is modified, or the at least one pair of guide RNAs is a pair of modified guide RNAs.

[0040] Embodiment 33 is the composition of any one of the preceding embodiments, wherein the one or more guide RNAs, the first and/or second guide RNA, the at least one pair of guide RNAs, or the nucleic acid is in a vector.

[0041] Embodiment 34 is the composition of embodiment 33, wherein the vector is a lipid nanoparticle or a liposome.

[0042] Embodiment 35 is the composition of embodiment 33, wherein the vector is a viral vector.

[0043] Embodiment 36 is the composition of embodiment 35, wherein the viral vector is an AAV vector.

[0044] Embodiment 37 is the composition of embodiment 36, wherein the AAV vector is an AAV9 vector.

[0045] Embodiment 38 is the composition of any one of the preceding embodiments, wherein a promoter for the one or more guide RNAs, the first and/or second guide RNA, or the at least one pair of guide RNAs is hU6c.

[0046] Embodiment 39 is the composition of any one of the preceding embodiments, wherein the one or more guide RNAs is a guide RNA for SluCas9, the first and/or second guide RNA is a guide RNA for SluCas9, or the at least one pair of guide RNAs is a pair of guide RNAs for SluCas9; and the one or more guide RNAs, the first and/or second guide RNA, or the at least one pair of guide RNAs comprise a scaffold comprising the sequence of any one of SEQ ID NOs: 600, 601, or 900-917.

[0047] Embodiment 40 is the composition of the immediately preceding embodiment, wherein the scaffold comprises the sequence of SEQ ID NO: 901. [0048] Embodiment 41 is the composition of any one of the preceding embodiments, wherein the one or more guide RNAs, the first and/or second guide RNA, or the at least one pair of guide RNAs is in an AAV vector, wherein the vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first guide RNA scaffold sequence; the reverse complement of a nucleic acid encoding a first guide RNA sequence; the reverse complement of a promoter for expression of the nucleic acid encoding the first guide RNA sequence; a promoter for expression of a nucleic acid encoding SluCas9; a nucleic acid encoding a SluCas9; a polyadenylation sequence; a promoter for expression of a second guide RNA sequence in the same direction as the promoter for SluCas9; a second RNA guide sequence; and a second guide RNA scaffold sequence.

[0049] Embodiment 42 is the composition of any one of the preceding embodiments, wherein the one or more guide RNAs, or the first and/or second guide RNA, is no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 nucleotides in length.

[0050] Embodiment 43 is a method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell the composition of any one of embodiments 1-42.

[0051] Embodiment 44 is a method of excising a DMD gene or a fragment of the DMD gene, the method comprising delivering to a cell the composition of any one of embodiments 1-42, wherein the size of the excised portion of the DMD gene is less than about 400 nucleotides.

[0052] Embodiment 45 is a composition comprising: a) a pair of guide RNA molecules or a nucleic acid encoding a pair of RNA molecules, wherein the first guide RNA targets a region upstream of a splice acceptor site or a splice donor site in a DMD gene and wherein the second guide RNA targets a region downstream of a splice acceptor site or a splice donor site in the DMD gene; and b) an endonuclease or a nucleic acid encoding an endonuclease, optionally wherein the endonuclease is a Staphylococcus lugdunensis Cas9 (SluCas9).

[0053] Embodiment 46 is the composition of embodiment 45, wherein the first guide RNA is in an intron of the DMD gene and the second guide RNA is in an exon of the DMD gene.

[0054] Embodiment 47 is the composition of embodiment 45, wherein the first guide RNA is in an exon of the DMD gene and the second guide RNA is in an intron of the DMD gene.

[0055] Embodiment 48 is the composition of embodiment 45, wherein the first guide RNA is in an exon of the DMD gene and the second guide RNA is in an exon of the DMD gene.

[0056] Embodiment 49 is the composition of embodiment 45, wherein the first guide RNA is in an intron of the DMD gene and the second guide RNA is in an intron of the DMD gene.

[0057] Embodiment 50 is the composition of any one of embodiments 45-49, wherein the splice acceptor site or splice donor site is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 78 of the DMD gene.

[0058] Embodiment 51 is the composition of any one of embodiments 45-50, wherein the splice acceptor site or splice donor site is in intron 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57 of the DMD gene.

[0059] Embodiment 52 is the composition of any one of embodiments 45-51, wherein the first guide RNA targets a region within 1-200, 1-150, 1-100, 1-50, 30-200, 30-150, 30-100, 30-50, 75-200, 75-150, 75-100, 100-200, 100-150, or 150-200 nucleotides upstream of the splice acceptor or splice donor site.

[0060] Embodiment 53 is the composition of any one of embodiments 45-52, wherein the first guide RNA targets a region within 1-200, 1-150, 1-100, 1-50, 30-200, 30-150, 30-100, 30-50, 75-200, 75-150, 75-100, 100-200, 100-150, or 150-200 nucleotides downstream of the splice acceptor or splice donor site.

[0061] Embodiment 54 is a method of excising a fragment of a DMD gene, the method comprising delivering to a cell the composition of any one of embodiments 45-53, wherein the pair of guide RNAs and the endonuclease excise a fragment of the DMD gene comprising the splice acceptor site or the splice donor site, wherein the fragment is between 3 and 400 nucleotides in length.

[0062] Embodiment 55 is the method of embodiment 54, wherein the excised fragment of the DMD gene comprises a splice acceptor site.

[0063] Embodiment 56 is the method of embodiment 54, wherein the excised fragment of the DMD gene comprises a splice donor site.

[0064] Embodiment 57 is the method of any one of embodiments 54-56, wherein the excised fragment is between 3-200, 3-150, 3-100, 3-50, 30-250, 30-200, 30-150, 30-100, 30-50, 75-250, 75- 200, 75-150, 75-100, 100-250, 100-200, 100-150, 150-250, 150-200, 200-250, 250-400, 300-400, or 350-400 nucleotides in length.

DESCRIPTION OF FIGURES

[0065] Figure 1 is a simplified schematic depicting the approximate excision fragment sizes for 24 tested guide pairs targeting intron 52 and intron 53 of the dystrophin gene, for excision of exon 53.

[0066] Figure 2 shows the editing frequency of selected SluCas9 guide pairs targeting intron 52 and intron 53 of the dystrophin gene, for excision of exon 53 in immortalized HsMM AE52 cells.

Each bar represents a sgRNA pair and the bar height depicts the average frequency of deletion of the sequence between the sgRNA pair targets, as determined by qPCR. Error bars denote the standard deviation for each sample group.

[0067] Figures 3A and 3B show dystrophin restoration in immortalized HsMM AE52 cells treated with selected SluCas9 sgRNA pairs. Figure 3A shows detection of dystrophin restoration in treated samples using a Jess protein assay. Figure 3B shows dystrophin restoration in immortalized HsMM AE52 cells treated with selected SluCas9 sgRNA pairs that result in either precise reframing deletion or deletion of the entire exon 53 of the dystrophin gene. Each bar represents a sgRNA pair and the bar height depicts normalized dystrophin protein signal of the sample as a percentage of normalized dystrophin expression in untreated WT13 cells. Error bars denote the standard deviation for each sample group. Samples treated with precise deletion or exon deletion pairs show approximately 30- 50% deletion.

[0068] Figure 4 is a set of three simplified schematics depicting guide pairing for three different editing goals: (top) exon deletion (e.g., deletion of exon 53 of the DMD gene using at least one guide pair selected from the guide pairs of Table 1D), (middle) splice acceptor deletion (e.g., skipping of exon 53 of the DMD gene using at least one guide pair selected from the guide pairs of Table IE), and (bottom) splice donor deletion (e.g., skipping of exon 53 of the DMD gene using at least one guide pair selected from the guide pairs of Table 1F).

[0069] Figure 5 shows dystrophin restoration in immortalized HsMM AE52 cells treated with selected SluCas9 or sRGN sgRNA pairs that result in either precise reframing deletion, deletion of the entire exon 53 of the dystrophin gene, or skipping of exon 53. Each bar represents a sgRNA pair or triplet and the bar height depicts dystrophin protein signal normalized to the total protein signal, as determined by Jess protein assay. All guide pairs were tested with SluCas9 with the exception of “sRGN3.3_96-103”, which was tested with sRGN3.3 and the E53SL96 and E53SL103 guides. Error bars denote the standard deviation for each sample group.

[0070] Figure 6 shows dystrophin restoration in immortalized HsMM AE52 cells treated with selected sgRNA pairs combined with SluCas9 or sRGN3.3 that result in either precise reframing deletion, deletion of the entire exon 53 of the dystrophin gene, or skipping of exon 53. Each bar represents a sgRNA pair and the bar height depicts dystrophin protein signal normalized to the total protein signal, as determined by Jess protein assay. All guide pairs were tested with SluCas9 with the exception of “[sRGN3.3]i52SL2_E53SL14”, which was tested with sRGN3.3 and the i52SL2 and E53SL14 guides. Error bars denote the standard deviation for each sample group.

DETAILED DESCRIPTION

[0071] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention is described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims and included embodiments.

[0072] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a guide” includes a plurality of guides and reference to “a cell” includes a plurality of cells and the like.

[0073] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0074] Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). The term “or” is used in an inclusive sense, i.e., equivalent to “and/or,” unless the context clearly indicates otherwise.

[0075] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

I. Definitions

[0076] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

[0077] “Polynucleotide,” “nucleic acid,” and “nucleic acid molecule,” are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2’ methoxy or 2’ halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1 -methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N⁴-methyl deoxyguanosine, deaza- or azapurines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5 -methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6- methylaminopurine, O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine -pyrimidines, and O⁴-alkyl-pyrimidines; US Pat. No. 5,378,825 and PCT No. WO 93/13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^th ed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2’ methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42): 13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

[0078] “Guide RNA” and simply “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” refers to each type. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences. For clarity, the terms “guide RNA” or “guide” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. In general, in the case of a DNA nucleic acid construct encoding a guide RNA, the U residues in any of the RNA sequences described herein may be replaced with T residues, and in the case of a guide RNA construct encoded by any of the DNA sequences described herein, the T residues may be replaced with U residues.

[0079] As used herein, a “spacer sequence,” sometimes also referred to herein and in the literature as a “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for cleavage by a Cas9. For clarity, the terms “spacer sequence”, “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. A guide sequence can be 24, 23, 22, 21, 20 or fewer base pairs in length, e.g., in the case of Staphylococcus lugdunensis (i.e., SluCas9) or Staphylococcus aureus (i.e., SaCas9) and related Cas9 homologs/orthologs. In preferred embodiments, a guide/spacer sequence in the case of SluCas9 or SaCas9 or sRGN is at least 20 base pairs in length, or more specifically, within 20-25 base pairs in length (see, e.g., Schmidt et al., 2021, Nature Communications, “Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases”). Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, or 25 -nucleotides in length. For example, in some embodiments, the guide sequence comprises at least 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-143. In some embodiments, the guide sequence comprises a sequence selected from SEQ ID NOs: 1-143. In some embodiments, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. For example, in some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-143. In some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 1-143. In preferred embodiments, a guide/spacer sequence in the case of SluCas9 or SaCas9 or a sRGN is at least 20 base pairs in length, or more specifically, within 20-25 base pairs in length see, e.g., Schmidt et al., 2021, Nature Communications, “Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases”). In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 16, 17, 18, 19, 20 or more base pairs. In some embodiments, the total length of the target sequence/guide is no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 22, no more than 23, or no more than 24 nucleotides in length. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides. In some embodiments, the guide sequence and the target region do not contain any mismatches.

[0080] In some embodiments, the guide sequence comprises a sequence selected from SEQ

ID NOs: 1-143, wherein if the 5’ terminal nucleotide is not guanine, one or more guanine (g) is added to the sequence at its 5’ end. In some embodiments, the 5’ g or gg is included in some instances for transcription, for example, for expression by the RNA polymerase Ill-dependent U6 promoter or the T7 promoter. In some embodiments, a 5’ guanine is added to any one of the guide sequences or pairs of guide sequences disclosed herein.

[0081] Target sequences for Cas9s include both the positive and negative strands of genomic

DNA (i.e., the sequence given and the sequence’s reverse complement), as a nucleic acid substrate for a Cas9 is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0082] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a guide RNA together with a Cas9. In some embodiments, the guide RNA guides the Cas9, such as a SluCas9 or a SaCas9, to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence, which can be followed by cleaving or nicking (in the context of a modified “nickase” Cas9).

[0083] As used herein, a first sequence is considered to “comprise a sequence with at least

X% identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X% or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5- methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5’-AXG where X is any modified uridine, such as pseudouridine, N1 -methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5’-CAU). Exemplary alignment algorithms are the Smith- Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate.

[0084] “mRNA” is used herein to refer to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2’ -methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2’ -methoxy ribose residues, or a combination thereof.

[0085] Guide sequences useful in the guide RNA compositions and methods described herein are shown, for example, in Tables 1A-1F, and throughout the specification.

[0086] As used herein, a “target sequence” refers to a sequence of nucleic acid in a target gene that has complementarity to at least a portion of the guide sequence of the guide RNA. The interaction of the target sequence and the guide sequence directs a Cas9 (such as a SluCas9) to bind, and potentially nick or cleave (depending on the activity of the agent), within or near the target sequence. A target sequence may be complementary to any of the guide RNA sequences disclosed herein (e.g., any of the guide RNA sequences disclosed in Tables 1A-1F).

[0087] As used herein, “treatment” refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease or development of the disease (which may occur before or after the disease is formally diagnosed, e.g., in cases where a subject has a genotype that has the potential or is likely to result in development of the disease), arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of DMD may comprise alleviating symptoms of DMD.

[0088] As used herein, “ameliorating” refers to any beneficial effect on a phenotype or symptom, such as reducing its severity, slowing or delaying its development, arresting its development, or partially or completely reversing or eliminating it. In the case of quantitative phenotypes such as expression levels, ameliorating encompasses changing the expression level so that it is closer to the expression level seen in healthy or unaffected cells or individuals.

[0089] A “pharmaceutically acceptable excipient” refers to an agent that is included in a pharmaceutical formulation that is not the active ingredient. Pharmaceutically acceptable excipients may e.g., aid in drug delivery or support or enhance stability or bioavailability.

[0090] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined.

[0091] As used herein, “Staphylococcus aureus Cas9” may also be referred to as SaCas9, and includes wild type SaCas9 (e.g., SEQ ID NO: 711) and variants thereof. A variant of SaCas9 comprises one or more amino acid changes as compared to SEQ ID NO: 711, including insertion, deletion, or substitution of one or more amino acids, or a chemical modification to one or more amino acids. For clarity, SaCas9KKH is a SaCas9 variant.

[0092] As used herein, “Staphylococcus lugdunensis Cas9” may also be referred to as

SluCas9, and includes wild type SluCas9 (e.g., SEQ ID NO: 712) and variants thereof. A variant of SluCas9 comprises one or more amino acid changes as compared to SEQ ID NO: 712, including insertion, deletion, or substitution of one or more amino acids, or a chemical modification to one or more amino acids.

[0093] As used herein, “splice acceptor site” refers to a portion of a gene necessary for splicing that includes a 3’ portion of an intron. Typically, a splice acceptor site comprises portion of a gene comprising at least the first two bases of a given exon (e.g., exon 53 of the dystrophin gene) and the last 26 bases of the upstream intron (e.g., intron 52 of the dystrophin gene). A sequence providing the dystrophin gene exon 53 splice acceptor site is provided in SEQ ID NO: 144, including nucleobases upstream and downstream of the splice acceptor site. In most mRNAs to be spliced, a splice acceptor site terminates an intron with an AG sequence.

[0094] As used herein, “splice donor site” refers to a portion of a gene necessary for splicing that includes a 5’ portion of an intron. Typically, a splice donor site comprises at least the three terminal nucleotides of a given exon (e.g., exon 53 of the dystrophin gene) and the first seven bases of the downstream intron (e.g., intron 53 of the dystrophin gene). A sequence providing the dystrophin gene exon 53 splice donor site is provided in SEQ ID NO: 145, including nucleobases upstream and downstream of the splice acceptor site. In most mRNAs to be spliced, a splice donor site includes a GU sequence at the 5' end of the intron (a GT sequence within the gene itself), within a larger, less highly conserved region.

[0095] As used here, “DMD gene” refers to the human gene (represented by Genbank

Accession No. NC_000023.11; SEQ ID NO: 146) encoding the dystrophin protein. Mutations (e.g., deletions, duplications, and/or point mutations) at the DMD gene locus have been associated with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy.

II. Compositions

[0096] Provided herein are compositions comprising guide RNAs and pairs of guide RNAs useful for treating Duchenne Muscular Dystrophy (DMD). The provided pairs of guide RNAs, when used with the correct endonuclease, function to excise all or a portion of exon 53 of the DMD gene. Tables 1A-1C provide a listing of guide sequences of guide RNAs, and Tables 1D-1F provide exemplary guide pairs.

Guides and Guide Pairs

A. Table 1A. Intron 52 SluCas9 guides: B. Table 1B. Exon 53 SluCas9 guides:

C. Table 1C. Intron 53 SluCas9 guides:

D. Table 1D. Intron 52-Intron 53 SluCas9 guide pairs:

E. Table 1E. Intron 52-Exon 53 SluCas9 guide pairs:

Ill

F. Table 1F. Exon 53-Intron 53 SluCas9 guide pairs:

Compositions

[0097] In some embodiments, a composition is provided comprising one or more guide RNAs, or one or more nucleic acids encoding one or more guide RNAs, comprising or consisting of one or more guide sequence of Tables 1A-1C.

[0098] In some embodiments, a composition is provided comprising one or more guide RNAs, or one or more nucleic acids encoding one or more guide RNAs, wherein the guide RNA comprises at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a guide sequence of Tables 1A-1C, or to a guide sequence comprising at least 16, 17, 18, 19, or 20 nucleotides of a guide sequence of Tables 1A-1C. In particular embodiments, a composition is provided comprising one or more guide RNAs, or one or more nucleic acids encoding one or more guide RNAs, wherein the guide RNA comprises at least 20 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a guide sequence comprising at least 20 nucleotides of a guide sequence of Tables 1A-1C. In other particular embodiments, a composition is provided comprising one or more guide RNAs, or one or more nucleic acids encoding one or more guide RNAs, wherein the guide RNA comprises no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to guide sequence comprising no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 contiguous nucleotides of a guide sequence of Tables 1A-1C.

[0099] In some embodiments, a composition is provided comprising two or more guide RNAs, or a nucleic acid encoding two or more guide RNAs, wherein each guide RNA comprises a guide sequence of Tables 1A-1C, or at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a guide sequence of Tables 1A-1C or to a guide sequence comprising at least 16, 17, 18, 19, or 20 nucleotides of a guide sequence of Tables 1A-1C. In particular embodiments, a composition is provided comprising two or more guide RNAs, or a nucleic acid encoding two or more guide RNAs, wherein each guide RNA comprises at least 20 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a guide sequence comprising at least 20 nucleotides of a guide sequence selected from Tables 1A-1C. In other particular embodiments, a composition is provided comprising two or more guide RNAs, or nucleic acid encoding two or more guide RNAs, wherein at least one of the two or more guide RNAs, optionally each guide RNA, comprises no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a guide sequence comprising no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 nucleotides of a guide sequence selected from Tables 1A-1C.

[00100] In some embodiments, a composition is provided comprising a pair of guide RNAs, or nucleic acid encoding a pair of guide RNAs, wherein the pair of guide RNAs comprises sequences at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a guide sequence to any of the pairs disclosed in Tables 1D-1F. In some embodiments, a composition is provided comprising a pair of guide RNAs, or nucleic acid encoding a pair of guide RNAs, wherein the pair of guide RNAs comprises sequences from any of the pairs disclosed in Tables 1D-1F. In some embodiments, a composition is provided comprising any of the pairs of guide RNA sequences disclosed in Tables 1D-1F.

[00101] In some embodiments, a composition is provided comprising one or more nucleic acid molecules, wherein at least one of the molecules comprises a nucleic acid encoding: a) a SluCas9; and b) a first and a second guide RNA comprising a first and a second guide sequence, wherein the first and second guide sequence are selected from any one of the guide sequence pairs of Tables 1D-1F.

[00102] In some embodiments, a composition is provided comprising two nucleic acid molecules, wherein at least one of the molecules comprises a nucleic acid encoding a first and a second guide RNA comprising a first and a second guide sequence, wherein the first and second guide sequence are selected from any one of the guide sequence pairs of Tables 1D-1F, optionally wherein the nucleic acid does not comprise a nucleic acid encoding an endonuclease.

[00103] In some embodiments, the composition further comprises an endonuclease or a nucleic acid encoding an endonuclease. An exemplary endonuclease for use with each of the guide RNAs or pairs of guide RNAs is provided herein, for example, the guide sequences provided in Tables 1A-1F are for use with a SluCas9 endonuclease. [00104] In some embodiments, a composition is provided comprising a single nucleic acid molecule comprising a nucleic acid encoding any of the guide RNAs disclosed herein, or any of the pairs of guide RNAs disclosed herein, and optionally a nucleic acid encoding an endonuclease.

[00105] In some embodiments, a composition is provided comprising at least two nucleic acid molecules comprising a nucleic acid encoding any of the guide RNAs disclosed herein, or any of the pairs of guide RNAs disclosed herein, and optionally a nucleic acid encoding an endonuclease, wherein at least one nucleic acid molecule does not comprise a nucleic acid encoding an endonuclease.

[00106] The disclosure herein may reference a “first and a second spacer” or a “first and a second guide RNA, gRNA, or sgRNA” followed by one or more pairs of specific sequences. It should be noted that the order of the sequences in the pair is not intended to be restricted to the order in which they are presented/described. For example, the phrase “the first sgRNA and the second sgRNA comprise the sequences of SEQ ID NOs: 10 and 15” could mean that the first sgRNA comprises the sequence of SEQ ID NO: 10 and the second sgRNA sequence comprises the sequence of SEQ ID NO: 15, or this phrase could mean that the first sgRNA comprises the sequence of SEQ ID NO: 15 and the second sgRNA sequence comprises the sequence of SEQ ID NO: 10.

[00107] In some embodiments, the first and/or the second nucleic acid, if present, comprises at least two guide RNAs. In some embodiments, the first and/or the second nucleic acid, if present, comprises at least three guide RNAs. In some embodiments, the first and/or the second nucleic acid, if present, comprises at least four guide RNAs. In some embodiments, the first and/or the second nucleic acid, if present, comprises at least five guide RNAs. In some embodiments, the first and/or the second nucleic acid, if present, comprises at least six guide RNAs. In some embodiments, the first nucleic acid encodes an endonuclease and at least one, at least two, or at least three guide RNAs. In some embodiments, the first nucleic acid comprises an endonuclease and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid. In some embodiments, the first nucleic acid encodes an endonuclease and from one to three guide RNAs. In some embodiments, the first nucleic acid comprises 1, 2, 3, 4, 5, or 6 guide RNAs, but does not encode an endonuclease.

[00108] In some embodiments, the second nucleic acid, if present, encodes at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs. In some embodiments, the second nucleic acid, if present, encodes from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid In some embodiments, the second nucleic acid, if present, encodes from one to six guide RNAs. In some embodiments, the second nucleic acid, if present, encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 guide RNAs. In some embodiments, the second nucleic acid, if present, encodes 2, 3, 4, 5, or 6 guide RNAs. In some embodiments, the second nucleic acid comprises 1, 2, 3, 4, 5, or 6 guide RNAs, but does not encode an endonuclease. [00109] In some embodiments, a single nucleic acid molecule comprises a nucleic acid encoding a Cas9 and a nucleic acid encoding two guide RNAs, wherein the nucleic acid molecule encodes no more than two guide RNAs.

[00110] In some embodiments, the single nucleic acid molecule is a single vector or comprised in a single vector. In some embodiments, the single vector expresses the two guide RNAs and Cas9. In some embodiments, a pair of guide RNAs and a Cas9 are provided on a single vector. In some embodiments, the single vector comprises a nucleic acid encoding a pair of guide RNAs and a nucleic acid encoding a SaCas9 or SluCas9. In some embodiments, two guide RNAs and a Cas9 are encoded on a single vector. In some embodiments, the single vector comprises a nucleic acid encoding a first guide RNA, a nucleic acid encoding a second guide RNA, and a nucleic acid encoding a SaCas9 or SluCas9.

[00111] In some embodiments, the first nucleic acid molecule encodes for a Cas9 molecule and also encodes for one or more copies of a first guide RNA and one or more copies of a second guide RNA. In some embodiments, the first nucleic acid molecule encodes for a Cas9 molecule, but does not encode for any guide RNAs. In some embodiments, the second nucleic acid molecule encodes for one or more copies of a first guide RNA and one or more copies of a second guide RNA, wherein the second nucleic acid molecule does not encode for a Cas9 molecule.

[00112] In some embodiments, any of the guides disclosed herein may be used as a research tool, e.g., to study trafficking, expression, and processing by cells.

A. Guide pairs targeting intron 52 and intron 53 of the DMD gene

[00113] In some embodiments, a composition is provided comprising a pair of guide RNAs, wherein the pair of guide RNAs comprises or consists of any one of the pairs of guide sequences of Table 1D. In some embodiments, a composition is provided comprising a pair of guide RNAs or a nucleic acid encoding a pair of guide RNAs, wherein the first guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 1-64; and the second guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 88-143. In some embodiments, a composition is provided comprising a pair of guide RNAs or a nucleic acid encoding a pair of guide RNAs, wherein the first guide RNA is selected from any one of SEQ ID NOs: 1-64; and the second guide RNA is selected from any one of SEQ ID NOs: 88-143. In some embodiments, the pair of guide RNAs is capable of excising at least a portion (such as all) of exon 53 of the DMD gene see, e.g., Figure 1 or Figure 4, top).

[00114] In some embodiments, a composition is provided comprising at least one pair of guide RNAs or a nucleic acid encoding at least one pair of guide RNAs, wherein the first and the second guide RNAs of the at least one pair of guide RNAs each comprise a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a first and a second guide sequence selected from any one of the following pairs of first and second guide sequences: a. SEQ ID NOs: 88 and 1; 88 and 2; 88 and 3; 88 and 4; 88 and 5; 88 and 6; 88 and 7; 88 and 8; 88 and 9; 88 and 10; 88 and 11; 88 and 12; 88 and 13; 88 and 14; 88 and 15; 88 and 16; 88 and 17; 88 and 18; 88 and 19; 88 and 20; 88 and 21; 88 and 22; 88 and 23; 88 and 24; 88 and 25; 88 and 26; 88 and 27; 88 and 28; 88 and 29; 88 and 30; 88 and 31; 88 and 32; 88 and 33; 88 and 34; 88 and 35; 88 and 36; 88 and 37; 88 and 38; 88 and 39; 88 and 40; 88 and 41; 88 and 42; 88 and 43; 88 and 44; 88 and 45; 88 and 46; 88 and 47; 88 and 48; 88 and 49; 88 and 50; 88 and 51; 88 and 52; 88 and 53; 88 and 54; 88 and 55; 88 and 56; 88 and 57; 88 and 58; 88 and 59; 88 and 60; 88 and 61; 88 and 62; 88 and 63; 88 and 64; 89 and 1; 89 and 2; 89 and 3; 89 and 4; 89 and 5; 89 and 6; 89 and 7; 89 and 8; 89 and 9; 89 and 10; 89 and 11; 89 and 12; 89 and 13; 89 and 14; 89 and 15; 89 and 16; 89 and 17; 89 and 18; 89 and 19; 89 and 20; 89 and 21; 89 and 22; 89 and 23; 89 and 24; 89 and 25; 89 and 26; 89 and 27; 89 and 28; 89 and 29; 89 and 30; 89 and 31; 89 and 32; 89 and 33; 89 and 34; 89 and 35; 89 and 36; 89 and 37; 89 and 38; 89 and 39; 89 and 40; 89 and 41; 89 and 42; 89 and 43; 89 and 44; 89 and 45; 89 and 46; 89 and 47; 89 and 48; 89 and 49; 89 and 50; 89 and 51; 89 and 52; 89 and 53; 89 and 54; 89 and 55; 89 and 56; 89 and 57; 89 and 58; 89 and 59; 89 and 60; 89 and 61; 89 and 62; 89 and 63; 89 and 64; 90 and 1; 90 and 2; 90 and 3; 90 and 4; 90 and 5; 90 and 6; 90 and 7; 90 and 8; 90 and 9; 90 and 10; 90 and 11; 90 and 12; 90 and 13; 90 and 14; 90 and 15; 90 and 16; 90 and 17; 90 and 18; 90 and 19; 90 and 20; 90 and 21; 90 and 22; 90 and 23; 90 and 24; 90 and 25; 90 and 26; 90 and 27; 90 and 28; 90 and 29; 90 and 30; 90 and 31; 90 and 32; 90 and 33; 90 and 34; 90 and 35; 90 and 36; 90 and 37; 90 and 38; 90 and 39; 90 and 40; 90 and 41; 90 and 42; 90 and 43; 90 and 44; 90 and 45; 90 and 46; 90 and 47; 90 and 48; 90 and 49; 90 and 50; 90 and 51; 90 and 52; 90 and 53; 90 and 54; 90 and 55; 90 and 56; 90 and 57; 90 and 58; 90 and 59; 90 and 60; 90 and 61; 90 and 62; 90 and 63; 90 and 64; 91 and 1; 91 and 2; 91 and 3; 91 and 4; 91 and 5; 91 and 6; 91 and 7; 91 and 8; 91 and 9; 91 and 10; 91 and 11; 91 and 12; 91 and 13; 91 and 14; 91 and 15; 91 and 16; 91 and 17; 91 and 18; 91 and 19; 91 and 20; 91 and 21; 91 and 22; 91 and 23; 91 and 24; 91 and 25; 91 and 26; 91 and 27; 91 and 28; 91 and 29; 91 and 30; 91 and 31; 91 and 32; 91 and 33; 91 and 34; 91 and 35; 91 and 36; 91 and 37; 91 and 38; 91 and 39; 91 and 40; 91 and 41; 91 and 42; 91 and 43; 91 and 44; 91 and 45; 91 and 46; 91 and 47; 91 and 48; 91 and 49; 91 and 50; 91 and 51; 91 and 52; 91 and 53; 91 and 54; 91 and 55; 91 and 56; 91 and 57; 91 and 58; 91 and 59; 91 and 60; 91 and 61; 91 and 62; 91 and 63; 91 and 64; 92 and 1; 92 and 2; 92 and 3; 92 and 4; 92 and 5; 92 and 6; 92 and 7; 92 and 8; 92 and 9; 92 and 10; 92 and 11; 92 and 12; 92 and 13; 92 and 14; 92 and 15; 92 and 16; 92 and 17; 92 and 18; 92 and 19; 92 and 20; 92 and 21; 92 and 22; 92 and 23; 92 and 24; 92 and 25; 92 and 26; 92 and 27; 92 and 28; 92 and 29; 92 and 30; 92 and 31; 92 and 32; 92 and 33; 92 and 34; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 92 and 43; 92 and 44; 92 and 45; 92 and 46; 92 and 47; 92 and 48; 92 and 49; 92 and 50; 92 and 51; 92 and 52; 92 and 53; 92 and 54; 92 and 55; 92 and 56; 92 and 57; 92 and 58; 92 and 59; 92 and 60; 92 and 61; 92 and 62; 92 and 63; 92 and 64; 93 and 1; 93 and 2; 93 and 3; 93 and 4; 93 and 5; 93 and 6; 93 and 7; 93 and 8; 93 and 9; 93 and 10; 93 and 11; 93 and 12; 93 and 13; 93 and 14; 93 and 15; 93 and 16; 93 and 17; 93 and 18; 93 and 19; 93 and 20; 93 and 21; 93 and 22; 93 and 23; 93 and 24; 93 and 25; 93 and 26; 93 and 27; 93 and 28; 93 and 29; 93 and 30; 93 and 31; 93 and 32; 93 and 33; 93 and 34; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; 93 and 42; 93 and 43; 93 and 44; 93 and 45; 93 and 46; 93 and 47; 93 and 48; 93 and 49; 93 and 50; 93 and 51; 93 and 52; 93 and 53; 93 and 54; 93 and 55; 93 and 56; 93 and 57; 93 and 58; 93 and 59; 93 and 60; 93 and 61; 93 and 62; 93 and 63; 93 and 64; 94 and 1; 94 and 2; 94 and 3; 94 and 4; 94 and 5; 94 and 6; 94 and 7; 94 and 8; 94 and 9; 94 and 10; 94 and 11; 94 and 12; 94 and 13; 94 and 14; 94 and 15; 94 and 16; 94 and 17; 94 and 18; 94 and 19; 94 and 20; 94 and 21; 94 and 22; 94 and 23; 94 and 24; 94 and 25; 94 and 26; 94 and 27; 94 and 28; 94 and 29; 94 and 30; 94 and 31; 94 and 32; 94 and 33; 94 and 34; 94 and 35; 94 and 36; 94 and 37; 94 and 38; 94 and 39; 94 and 40; 94 and 41; 94 and 42; 94 and 43; 94 and 44; 94 and 45; 94 and 46; 94 and 47; 94 and 48; 94 and 49; 94 and 50; 94 and 51; 94 and 52; 94 and 53; 94 and 54; 94 and 55; 94 and 56; 94 and 57; 94 and 58; 94 and 59; 94 and 60; 94 and 61; 94 and 62; 94 and 63; 94 and 64; 95 and 1; 95 and 2; 95 and 3; 95 and 4; 95 and 5; 95 and 6; 95 and 7; 95 and 8; 95 and 9; 95 and 10; 95 and 11; 95 and 12; 95 and 13; 95 and 14; 95 and 15; 95 and 16; 95 and 17; 95 and 18; 95 and 19; 95 and 20; 95 and 21; 95 and 22; 95 and 23; 95 and 24; 95 and 25; 95 and 26; 95 and 27; 95 and 28; 95 and 29; 95 and 30; 95 and 31; 95 and 32; 95 and 33; 95 and 34; 95 and 35; 95 and 36; 95 and 37; 95 and 38; 95 and 39; 95 and 40; 95 and 41; 95 and 42; 95 and 43; 95 and 44; 95 and 45; 95 and 46; 95 and 47; 95 and 48; 95 and 49; 95 and 50; 95 and 51; 95 and 52; 95 and 53; 95 and 54; 95 and 55; 95 and 56; 95 and 57; 95 and 58; 95 and 59; 95 and 60; 95 and 61; 95 and 62; 95 and 63; 95 and 64; 96 and 1; 96 and 2; 96 and 3; 96 and 4; 96 and 5; 96 and 6; 96 and 7; 96 and 8; 96 and 9; 96 and 10; 96 and 11; 96 and 12; 96 and 13; 96 and 14; 96 and 15; 96 and 16; 96 and 17; 96 and 18; 96 and 19; 96 and 20; 96 and 21; 96 and 22; 96 and 23; 96 and 24; 96 and 25; 96 and 26; 96 and 27; 96 and 28; 96 and 29; 96 and 30; 96 and 31; 96 and 32; 96 and 33; 96 and 34; 96 and 35; 96 and 36; 96 and 37; 96 and 38; 96 and 39; 96 and 40; 96 and 41; 96 and 42; 96 and 43; 96 and 44; 96 and 45; 96 and 46; 96 and 47; 96 and 48; 96 and 49; 96 and 50; 96 and 51; 96 and 52; 96 and 53; 96 and 54; 96 and 55; 96 and 56; 96 and 57; 96 and 58; 96 and 59; 96 and 60; 96 and 61; 96 and 62; 96 and 63; 96 and 64; 97 and 1; 97 and 2; 97 and 3; 97 and 4; 97 and 5; 97 and 6; 97 and 7; 97 and 8; 97 and 9; 97 and 10; 97 and 11; 97 and 12; 97 and 13; 97 and 14; 97 and 15; 97 and 16; 97 and 17; 97 and 18; 97 and 19; 97 and 20; 97 and 21; 97 and 22; 97 and 23; 97 and 24; 97 and 25; 97 and 26; 97 and 27; 97 and 28; 97 and 29; 97 and 30; 97 and 31; 97 and 32; 97 and 33; 97 and 34; 97 and 35; 97 and 36; 97 and 37; 97 and 38; 97 and 39; 97 and 40; 97 and 41; 97 and 42; 97 and 43; 97 and 44; 97 and 45; 97 and 46; 97 and 47; 97 and 48; 97 and 49; 97 and 50; 97 and 51; 97 and 52; 97 and 53; 97 and 54; 97 and 55; 97 and 56; 97 and 57; 97 and 58; 97 and 59; 97 and 60; 97 and 61; 97 and 62; 97 and 63; 97 and 64; 98 and 1; 98 and 2; 98 and 3; 98 and 4; 98 and 5; 98 and 6; 98 and 7; 98 and 8; 98 and 9; 98 and 10; 98 and 11; 98 and 12; 98 and 13; 98 and 14; 98 and 15; 98 and 16; 98 and 17; 98 and 18; 98 and 19; 98 and 20; 98 and 21; 98 and 22; 98 and 23; 98 and 24; 98 and 25; 98 and 26; 98 and 27; 98 and 28; 98 and 29; 98 and 30; 98 and 31; 98 and 32; 98 and 33; 98 and 34; 98 and 35; 98 and 36; 98 and 37; 98 and 38; 98 and 39; 98 and 40; 98 and 41; 98 and 42; 98 and 43; 98 and 44; 98 and 45; 98 and 46; 98 and 47; 98 and 48; 98 and 49; 98 and 50; 98 and 51; 98 and 52; 98 and 53; 98 and 54; 98 and 55; 98 and 56; 98 and 57; 98 and 58; 98 and 59; 98 and 60; 98 and 61; 98 and 62; 98 and 63; 98 and 64; 99 and 1; 99 and 2; 99 and 3; 99 and 4; 99 and 5; 99 and 6; 99 and 7; 99 and 8; 99 and 9; 99 and 10; 99 and 11; 99 and 12; 99 and 13; 99 and 14; 99 and 15; 99 and 16; 99 and 17; 99 and 18; 99 and 19; 99 and 20; 99 and 21; 99 and 22; 99 and 23; 99 and 24; 99 and 25; 99 and 26; 99 and 27; 99 and 28; 99 and 29; 99 and 30; 99 and 31; 99 and 32; 99 and 33; 99 and 34; 99 and 35; 99 and 36; 99 and 37; 99 and 38; 99 and 39; 99 and 40; 99 and 41; 99 and 42; 99 and 43; 99 and 44; 99 and 45; 99 and 46; 99 and 47; 99 and 48; 99 and 49; 99 and 50; 99 and 51; 99 and 52; 99 and 53; 99 and 54; 99 and 55; 99 and 56; 99 and 57; 99 and 58; 99 and 59; 99 and 60; 99 and 61; 99 and 62; 99 and 63; 99 and 64; 100 and 1; 100 and 2; 100 and 3; 100 and 4; 100 and 5; 100 and 6; 100 and 7; 100 and 8; 100 and 9; 100 and 10; 100 and 11; 100 and 12; 100 and 13; 100 and 14; 100 and 15; 100 and 16; 100 and 17; 100 and 18; 100 and 19; 100 and 20; 100 and 21; 100 and 22; 100 and 23; 100 and 24; 100 and 25; 100 and 26; 100 and 27; 100 and 28; 100 and 29; 100 and 30; 100 and 31; 100 and 32; 100 and 33; 100 and 34; 100 and 35; 100 and 36; 100 and 37; 100 and 38; 100 and 39; 100 and 40; 100 and 41; 100 and 42; 100 and 43; 100 and 44; 100 and 45; 100 and 46; 100 and 47; 100 and 48; 100 and 49; 100 and 50; 100 and 51; 100 and 52; 100 and 53; 100 and 54; 100 and 55; 100 and 56; 100 and 57; 100 and 58; 100 and 59; 100 and 60; 100 and 61; 100 and 62;

100 and 63; 100 and 64; 101 and 1; 101 and 2; 101 and 3; 101 and 4; 101 and 5; 101 and 6; 101 and 7; 101 and 8; 101 and 9; 101 and 10; 101 and 11; 101 and 12; 101 and 13; 101 and 14; 101 and 15; 101 and 16; 101 and 17; 101 and 18; 101 and 19; 101 and 20; 101 and 21; 101 and 22;

101 and 23; 101 and 24; 101 and 25; 101 and 26; 101 and 27; 101 and 28; 101 and 29; 101 and 30; 101 and 31; 101 and 32; 101 and 33; 101 and 34; 101 and 35; 101 and 36; 101 and 37; 101 and 38; 101 and 39; 101 and 40; 101 and 41; 101 and 42; 101 and 43; 101 and 44; 101 and 45; 101 and 46; 101 and 47; 101 and 48; 101 and 49; 101 and 50; 101 and 51; 101 and 52; 101 and 53; 101 and 54; 101 and 55; 101 and 56; 101 and 57; 101 and 58; 101 and 59; 101 and 60; 101 and 61; 101 and 62; 101 and 63; 101 and 64; 102 and 1; 102 and 2; 102 and 3; 102 and 4; 102 and 5; 102 and 6; 102 and 7; 102 and 8; 102 and 9; 102 and 10; 102 and 11; 102 and 12; 102 and 13; 102 and 14; 102 and 15; 102 and 16; 102 and 17; 102 and 18; 102 and 19; 102 and 20; 102 and 21; 102 and 22; 102 and 23; 102 and 24; 102 and 25; 102 and 26; 102 and 27; 102 and 28; 102 and 29; 102 and 30; 102 and 31; 102 and 32; 102 and 33; 102 and 34; 102 and 35; 102 and 36; 102 and 37; 102 and 38; 102 and 39; 102 and 40; 102 and 41; 102 and 42; 102 and 43;

102 and 44; 102 and 45; 102 and 46; 102 and 47; 102 and 48; 102 and 49; 102 and 50; 102 and 51; 102 and 52; 102 and 53; 102 and 54; 102 and 55; 102 and 56; 102 and 57; 102 and 58; 102 and 59; 102 and 60; 102 and 61; 102 and 62; 102 and 63; 102 and 64; 103 and 1; 103 and 2; 103 and 3; 103 and 4; 103 and 5; 103 and 6; 103 and 7; 103 and 8; 103 and 9; 103 and 10; 103 and 11; 103 and 12; 103 and 13; 103 and 14; 103 and 15; 103 and 16; 103 and 17; 103 and 18; 103 and 19; 103 and 20; 103 and 21; 103 and 22; 103 and 23; 103 and 24; 103 and 25; 103 and 26;

103 and 27; 103 and 28; 103 and 29; 103 and 30; 103 and 31; 103 and 32; 103 and 33; 103 and 34; 103 and 35; 103 and 36; 103 and 37; 103 and 38; 103 and 39; 103 and 40; 103 and 41; 103 and 42; 103 and 43; 103 and 44; 103 and 45; 103 and 46; 103 and 47; 103 and 48; 103 and 49;

103 and 50; 103 and 51; 103 and 52; 103 and 53; 103 and 54; 103 and 55; 103 and 56; 103 and 57; 103 and 58; 103 and 59; 103 and 60; 103 and 61; 103 and 62; 103 and 63; 103 and 64; 104 and 1; 104 and 2; 104 and 3; 104 and 4; 104 and 5; 104 and 6; 104 and 7; 104 and 8; 104 and 9;

104 and 10; 104 and 11; 104 and 12; 104 and 13; 104 and 14; 104 and 15; 104 and 16; 104 and 17; 104 and 18; 104 and 19; 104 and 20; 104 and 21; 104 and 22; 104 and 23; 104 and 24; 104 and 25; 104 and 26; 104 and 27; 104 and 28; 104 and 29; 104 and 30; 104 and 31; 104 and 32; 104 and 33; 104 and 34; 104 and 35; 104 and 36; 104 and 37; 104 and 38; 104 and 39; 104 and 40; 104 and 41; 104 and 42; 104 and 43; 104 and 44; 104 and 45; 104 and 46; 104 and 47; 104 and 48; 104 and 49; 104 and 50; 104 and 51; 104 and 52; 104 and 53; 104 and 54; 104 and 55;

104 and 56; 104 and 57; 104 and 58; 104 and 59; 104 and 60; 104 and 61; 104 and 62; 104 and 63; 104 and 64; 105 and 1; 105 and 2; 105 and 3; 105 and 4; 105 and 5; 105 and 6; 105 and 7;

105 and 8; 105 and 9; 105 and 10; 105 and 11; 105 and 12; 105 and 13; 105 and 14; 105 and 15; 105 and 16; 105 and 17; 105 and 18; 105 and 19; 105 and 20; 105 and 21; 105 and 22; 105 and 23; 105 and 24; 105 and 25; 105 and 26; 105 and 27; 105 and 28; 105 and 29; 105 and 30; 105 and 31; 105 and 32; 105 and 33; 105 and 34; 105 and 35; 105 and 36; 105 and 37; 105 and 38; 105 and 39; 105 and 40; 105 and 41; 105 and 42; 105 and 43; 105 and 44; 105 and 45; 105 and 46; 105 and 47; 105 and 48; 105 and 49; 105 and 50; 105 and 51; 105 and 52; 105 and 53; 105 and 54; 105 and 55; 105 and 56; 105 and 57; 105 and 58; 105 and 59; 105 and 60; 105 and 61;

105 and 62; 105 and 63; 105 and 64; 106 and 1; 106 and 2; 106 and 3; 106 and 4; 106 and 5;

106 and 6; 106 and 7; 106 and 8; 106 and 9; 106 and 10; 106 and 11; 106 and 12; 106 and 13; 106 and 14; 106 and 15; 106 and 16; 106 and 17; 106 and 18; 106 and 19; 106 and 20; 106 and 21; 106 and 22; 106 and 23; 106 and 24; 106 and 25; 106 and 26; 106 and 27; 106 and 28; 106 and 29; 106 and 30; 106 and 31; 106 and 32; 106 and 33; 106 and 34; 106 and 35; 106 and 36; 106 and 37; 106 and 38; 106 and 39; 106 and 40; 106 and 41; 106 and 42; 106 and 43; 106 and 44; 106 and 45; 106 and 46; 106 and 47; 106 and 48; 106 and 49; 106 and 50; 106 and 51; 106 and 52; 106 and 53; 106 and 54; 106 and 55; 106 and 56; 106 and 57; 106 and 58; 106 and 59;

106 and 60; 106 and 61; 106 and 62; 106 and 63; 106 and 64; 107 and 1; 107 and 2; 107 and 3;

107 and 4; 107 and 5; 107 and 6; 107 and 7; 107 and 8; 107 and 9; 107 and 10; 107 and 11; 107 and 12; 107 and 13; 107 and 14; 107 and 15; 107 and 16; 107 and 17; 107 and 18; 107 and 19; 107 and 20; 107 and 21; 107 and 22; 107 and 23; 107 and 24; 107 and 25; 107 and 26; 107 and 27; 107 and 28; 107 and 29; 107 and 30; 107 and 31; 107 and 32; 107 and 33; 107 and 34; 107 and 35; 107 and 36; 107 and 37; 107 and 38; 107 and 39; 107 and 40; 107 and 41; 107 and 42;

107 and 43; 107 and 44; 107 and 45; 107 and 46; 107 and 47; 107 and 48; 107 and 49; 107 and 50; 107 and 51; 107 and 52; 107 and 53; 107 and 54; 107 and 55; 107 and 56; 107 and 57; 107 and 58; 107 and 59; 107 and 60; 107 and 61; 107 and 62; 107 and 63; 107 and 64; 108 and 1;

108 and 2; 108 and 3; 108 and 4; 108 and 5; 108 and 6; 108 and 7; 108 and 8; 108 and 9; 108 and 10; 108 and 11; 108 and 12; 108 and 13; 108 and 14; 108 and 15; 108 and 16; 108 and 17; 108 and 18; 108 and 19; 108 and 20; 108 and 21; 108 and 22; 108 and 23; 108 and 24; 108 and 25; 108 and 26; 108 and 27; 108 and 28; 108 and 29; 108 and 30; 108 and 31; 108 and 32; 108 and 33; 108 and 34; 108 and 35; 108 and 36; 108 and 37; 108 and 38; 108 and 39; 108 and 40; 108 and 41; 108 and 42; 108 and 43; 108 and 44; 108 and 45; 108 and 46; 108 and 47; 108 and 48; 108 and 49; 108 and 50; 108 and 51; 108 and 52; 108 and 53; 108 and 54; 108 and 55; 108 and 56; 108 and 57; 108 and 58; 108 and 59; 108 and 60; 108 and 61; 108 and 62; 108 and 63;

108 and 64; 109 and 1; 109 and 2; 109 and 3; 109 and 4; 109 and 5; 109 and 6; 109 and 7; 109 and 8; 109 and 9; 109 and 10; 109 and 11; 109 and 12; 109 and 13; 109 and 14; 109 and 15; 109 and 16; 109 and 17; 109 and 18; 109 and 19; 109 and 20; 109 and 21; 109 and 22; 109 and 23;

109 and 24; 109 and 25; 109 and 26; 109 and 27; 109 and 28; 109 and 29; 109 and 30; 109 and 31; 109 and 32; 109 and 33; 109 and 34; 109 and 35; 109 and 36; 109 and 37; 109 and 38; 109 and 39; 109 and 40; 109 and 41; 109 and 42; 109 and 43; 109 and 44; 109 and 45; 109 and 46;

109 and 47; 109 and 48; 109 and 49; 109 and 50; 109 and 51; 109 and 52; 109 and 53; 109 and 54; 109 and 55; 109 and 56; 109 and 57; 109 and 58; 109 and 59; 109 and 60; 109 and 61; 109 and 62; 109 and 63; 109 and 64; 110 and 1; 110 and 2; 110 and 3; 110 and 4; 110 and 5; 110 and 6; 110 and 7; 110 and 8; 110 and 9; 110 and 10; 110 and 11; 110 and 12; 110 and 13; 110 and 14; 110 and 15; 110 and 16; 110 and 17; 110 and 18; 110 and 19; 110 and 20; 110 and 21;

110 and 22; 110 and 23; 110 and 24; 110 and 25; 110 and 26; 110 and 27; 110 and 28; 110 and 29; 110 and 30; 110 and 31; 110 and 32; 110 and 33; 110 and 34; 110 and 35; 110 and 36; 110 and 37; 110 and 38; 110 and 39; 110 and 40; 110 and 41; 110 and 42; 110 and 43; 110 and 44; 110 and 45; 110 and 46; 110 and 47; 110 and 48; 110 and 49; 110 and 50; 110 and 51; 110 and 52; 110 and 53; 110 and 54; 110 and 55; 110 and 56; 110 and 57; 110 and 58; 110 and 59; 110 and 60; 110 and 61; 110 and 62; 110 and 63; 110 and 64; 111 and 1; 111 and 2; 111 and 3; 111 and 4; 111 and 5; 111 and 6; 111 and 7; 111 and 8; 111 and 9; 111 and 10; 111 and 11; 111 and 12; 111 and 13; 111 and 14; 111 and 15; 111 and 16; 111 and 17; 111 and 18; 111 and 19; 111 and 20; 111 and 21; 111 and 22; 111 and 23; 111 and 24; 111 and 25; 111 and 26; 111 and 27; 111 and 28; 111 and 29; 111 and 30; 111 and 31; 111 and 32; 111 and 33; 111 and 34; 111 and 35; 111 and 36; 111 and 37; 111 and 38; 111 and 39; 111 and 40; 111 and 41; 111 and 42; 111 and 43; 111 and 44; 111 and 45; 111 and 46; 111 and 47; 111 and 48; 111 and 49; 111 and 50;

111 and 51; 111 and 52; 111 and 53; 111 and 54; 111 and 55; 111 and 56; 111 and 57; 111 and 58; 111 and 59; 111 and 60; 111 and 61; 111 and 62; 111 and 63; 111 and 64; 112 and 1; 112 and 2; 112 and 3; 112 and 4; 112 and 5; 112 and 6; 112 and 7; 112 and 8; 112 and 9; 112 and 10; 112 and 11; 112 and 12; 112 and 13; 112 and 14; 112 and 15; 112 and 16; 112 and 17; 112 and 18; 112 and 19; 112 and 20; 112 and 21; 112 and 22; 112 and 23; 112 and 24; 112 and 25;

112 and 26; 112 and 27; 112 and 28; 112 and 29; 112 and 30; 112 and 31; 112 and 32; 112 and 33; 112 and 34; 112 and 35; 112 and 36; 112 and 37; 112 and 38; 112 and 39; 112 and 40; 112 and 41; 112 and 42; 112 and 43; 112 and 44; 112 and 45; 112 and 46; 112 and 47; 112 and 48;

112 and 49; 112 and 50; 112 and 51; 112 and 52; 112 and 53; 112 and 54; 112 and 55; 112 and 56; 112 and 57; 112 and 58; 112 and 59; 112 and 60; 112 and 61; 112 and 62; 112 and 63; 112 and 64; 113 and 1; 113 and 2; 113 and 3; 113 and 4; 113 and 5; 113 and 6; 113 and 7; 113 and 8; 113 and 9; 113 and 10; 113 and 11; 113 and 12; 113 and 13; 113 and 14; 113 and 15; 113 and 16; 113 and 17; 113 and 18; 113 and 19; 113 and 20; 113 and 21; 113 and 22; 113 and 23; 113 and 24; 113 and 25; 113 and 26; 113 and 27; 113 and 28; 113 and 29; 113 and 30; 113 and 31;

113 and 32; 113 and 33; 113 and 34; 113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 43; 113 and 44; 113 and 45; 113 and 46; 113 and 47; 113 and 48; 113 and 49; 113 and 50; 113 and 51; 113 and 52; 113 and 53; 113 and 54;

113 and 55; 113 and 56; 113 and 57; 113 and 58; 113 and 59; 113 and 60; 113 and 61; 113 and 62; 113 and 63; 113 and 64; 114 and 1; 114 and 2; 114 and 3; 114 and 4; 114 and 5; 114 and 6;

114 and 7; 114 and 8; 114 and 9; 114 and 10; 114 and 11; 114 and 12; 114 and 13; 114 and 14; 114 and 15; 114 and 16; 114 and 17; 114 and 18; 114 and 19; 114 and 20; 114 and 21; 114 and 22; 114 and 23; 114 and 24; 114 and 25; 114 and 26; 114 and 27; 114 and 28; 114 and 29; 114 and 30; 114 and 31; 114 and 32; 114 and 33; 114 and 34; 114 and 35; 114 and 36; 114 and 37; 114 and 38; 114 and 39; 114 and 40; 114 and 41; 114 and 42; 114 and 43; 114 and 44; 114 and 45; 114 and 46; 114 and 47; 114 and 48; 114 and 49; 114 and 50; 114 and 51; 114 and 52; 114 and 53; 114 and 54; 114 and 55; 114 and 56; 114 and 57; 114 and 58; 114 and 59; 114 and 60;

114 and 61; 114 and 62; 114 and 63; 114 and 64; 115 and 1; 115 and 2; 115 and 3; 115 and 4;

115 and 5; 115 and 6; 115 and 7; 115 and 8; 115 and 9; 115 and 10; 115 and 11; 115 and 12; 115 and 13; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 18; 115 and 19; 115 and 20; 115 and 21; 115 and 22; 115 and 23; 115 and 24; 115 and 25; 115 and 26; 115 and 27; 115 and 28; 115 and 29; 115 and 30; 115 and 31; 115 and 32; 115 and 33; 115 and 34; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 43; 115 and 44; 115 and 45; 115 and 46; 115 and 47; 115 and 48; 115 and 49; 115 and 50; 115 and 51; 115 and 52; 115 and 53; 115 and 54; 115 and 55; 115 and 56; 115 and 57; 115 and 58;

115 and 59; 115 and 60; 115 and 61; 115 and 62; 115 and 63; 115 and 64; 116 and 1; 116 and 2;

116 and 3; 116 and 4; 116 and 5; 116 and 6; 116 and 7; 116 and 8; 116 and 9; 116 and 10; 116 and 11; 116 and 12; 116 and 13; 116 and 14; 116 and 15; 116 and 16; 116 and 17; 116 and 18; 116 and 19; 116 and 20; 116 and 21; 116 and 22; 116 and 23; 116 and 24; 116 and 25; 116 and 26; 116 and 27; 116 and 28; 116 and 29; 116 and 30; 116 and 31; 116 and 32; 116 and 33; 116 and 34; 116 and 35; 116 and 36; 116 and 37; 116 and 38; 116 and 39; 116 and 40; 116 and 41;

116 and 42; 116 and 43; 116 and 44; 116 and 45; 116 and 46; 116 and 47; 116 and 48; 116 and 49; 116 and 50; 116 and 51; 116 and 52; 116 and 53; 116 and 54; 116 and 55; 116 and 56; 116 and 57; 116 and 58; 116 and 59; 116 and 60; 116 and 61; 116 and 62; 116 and 63; 116 and 64;

117 and 1; 117 and 2; 117 and 3; 117 and 4; 117 and 5; 117 and 6; 117 and 7; 117 and 8; 117 and 9; 117 and 10; 117 and 11; 117 and 12; 117 and 13; 117 and 14; 117 and 15; 117 and 16; 117 and 17; 117 and 18; 117 and 19; 117 and 20; 117 and 21; 117 and 22; 117 and 23; 117 and 24; 117 and 25; 117 and 26; 117 and 27; 117 and 28; 117 and 29; 117 and 30; 117 and 31; 117 and 32; 117 and 33; 117 and 34; 117 and 35; 117 and 36; 117 and 37; 117 and 38; 117 and 39; 117 and 40; 117 and 41; 117 and 42; 117 and 43; 117 and 44; 117 and 45; 117 and 46; 117 and 47; 117 and 48; 117 and 49; 117 and 50; 117 and 51; 117 and 52; 117 and 53; 117 and 54; 117 and 55; 117 and 56; 117 and 57; 117 and 58; 117 and 59; 117 and 60; 117 and 61; 117 and 62;

117 and 63; 117 and 64; 118 and 1; 118 and 2; 118 and 3; 118 and 4; 118 and 5; 118 and 6; 118 and 7; 118 and 8; 118 and 9; 118 and 10; 118 and 11; 118 and 12; 118 and 13; 118 and 14; 118 and 15; 118 and 16; 118 and 17; 118 and 18; 118 and 19; 118 and 20; 118 and 21; 118 and 22;

118 and 23; 118 and 24; 118 and 25; 118 and 26; 118 and 27; 118 and 28; 118 and 29; 118 and 30; 118 and 31; 118 and 32; 118 and 33; 118 and 34; 118 and 35; 118 and 36; 118 and 37; 118 and 38; 118 and 39; 118 and 40; 118 and 41; 118 and 42; 118 and 43; 118 and 44; 118 and 45;

118 and 46; 118 and 47; 118 and 48; 118 and 49; 118 and 50; 118 and 51; 118 and 52; 118 and 53; 118 and 54; 118 and 55; 118 and 56; 118 and 57; 118 and 58; 118 and 59; 118 and 60; 118 and 61; 118 and 62; 118 and 63; 118 and 64; 119 and 1; 119 and 2; 119 and 3; 119 and 4; 119 and 5; 119 and 6; 119 and 7; 119 and 8; 119 and 9; 119 and 10; 119 and 11; 119 and 12; 119 and 13; 119 and 14; 119 and 15; 119 and 16; 119 and 17; 119 and 18; 119 and 19; 119 and 20;

119 and 21; 119 and 22; 119 and 23; 119 and 24; 119 and 25; 119 and 26; 119 and 27; 119 and 28; 119 and 29; 119 and 30; 119 and 31; 119 and 32; 119 and 33; 119 and 34; 119 and 35; 119 and 36; 119 and 37; 119 and 38; 119 and 39; 119 and 40; 119 and 41; 119 and 42; 119 and 43; 119 and 44; 119 and 45; 119 and 46; 119 and 47; 119 and 48; 119 and 49; 119 and 50; 119 and 51; 119 and 52; 119 and 53; 119 and 54; 119 and 55; 119 and 56; 119 and 57; 119 and 58; 119 and 59; 119 and 60; 119 and 61; 119 and 62; 119 and 63; 119 and 64; 120 and 1; 120 and 2; 120 and 3; 120 and 4; 120 and 5; 120 and 6; 120 and 7; 120 and 8; 120 and 9; 120 and 10; 120 and 11; 120 and 12; 120 and 13; 120 and 14; 120 and 15; 120 and 16; 120 and 17; 120 and 18; 120 and 19; 120 and 20; 120 and 21; 120 and 22; 120 and 23; 120 and 24; 120 and 25; 120 and 26; 120 and 27; 120 and 28; 120 and 29; 120 and 30; 120 and 31; 120 and 32; 120 and 33; 120 and 34; 120 and 35; 120 and 36; 120 and 37; 120 and 38; 120 and 39; 120 and 40; 120 and 41; 120 and 42; 120 and 43; 120 and 44; 120 and 45; 120 and 46; 120 and 47; 120 and 48; 120 and 49;

120 and 50; 120 and 51; 120 and 52; 120 and 53; 120 and 54; 120 and 55; 120 and 56; 120 and 57; 120 and 58; 120 and 59; 120 and 60; 120 and 61; 120 and 62; 120 and 63; 120 and 64; 121 and 1; 121 and 2; 121 and 3; 121 and 4; 121 and 5; 121 and 6; 121 and 7; 121 and 8; 121 and 9;

121 and 10; 121 and 11; 121 and 12; 121 and 13; 121 and 14; 121 and 15; 121 and 16; 121 and 17; 121 and 18; 121 and 19; 121 and 20; 121 and 21; 121 and 22; 121 and 23; 121 and 24; 121 and 25; 121 and 26; 121 and 27; 121 and 28; 121 and 29; 121 and 30; 121 and 31; 121 and 32; 121 and 33; 121 and 34; 121 and 35; 121 and 36; 121 and 37; 121 and 38; 121 and 39; 121 and 40; 121 and 41; 121 and 42; 121 and 43; 121 and 44; 121 and 45; 121 and 46; 121 and 47; 121 and 48; 121 and 49; 121 and 50; 121 and 51; 121 and 52; 121 and 53; 121 and 54; 121 and 55;

121 and 56; 121 and 57; 121 and 58; 121 and 59; 121 and 60; 121 and 61; 121 and 62; 121 and 63; 121 and 64; 122 and 1; 122 and 2; 122 and 3; 122 and 4; 122 and 5; 122 and 6; 122 and 7;

122 and 8; 122 and 9; 122 and 10; 122 and 11; 122 and 12; 122 and 13; 122 and 14; 122 and 15; 122 and 16; 122 and 17; 122 and 18; 122 and 19; 122 and 20; 122 and 21; 122 and 22; 122 and 23; 122 and 24; 122 and 25; 122 and 26; 122 and 27; 122 and 28; 122 and 29; 122 and 30; 122 and 31; 122 and 32; 122 and 33; 122 and 34; 122 and 35; 122 and 36; 122 and 37; 122 and 38; 122 and 39; 122 and 40; 122 and 41; 122 and 42; 122 and 43; 122 and 44; 122 and 45; 122 and 46; 122 and 47; 122 and 48; 122 and 49; 122 and 50; 122 and 51; 122 and 52; 122 and 53; 122 and 54; 122 and 55; 122 and 56; 122 and 57; 122 and 58; 122 and 59; 122 and 60; 122 and 61;

122 and 62; 122 and 63; 122 and 64; 123 and 1; 123 and 2; 123 and 3; 123 and 4; 123 and 5;

123 and 6; 123 and 7; 123 and 8; 123 and 9; 123 and 10; 123 and 11; 123 and 12; 123 and 13; 123 and 14; 123 and 15; 123 and 16; 123 and 17; 123 and 18; 123 and 19; 123 and 20; 123 and 21; 123 and 22; 123 and 23; 123 and 24; 123 and 25; 123 and 26; 123 and 27; 123 and 28; 123 and 29; 123 and 30; 123 and 31; 123 and 32; 123 and 33; 123 and 34; 123 and 35; 123 and 36; 123 and 37; 123 and 38; 123 and 39; 123 and 40; 123 and 41; 123 and 42; 123 and 43; 123 and 44; 123 and 45; 123 and 46; 123 and 47; 123 and 48; 123 and 49; 123 and 50; 123 and 51; 123 and 52; 123 and 53; 123 and 54; 123 and 55; 123 and 56; 123 and 57; 123 and 58; 123 and 59;

123 and 60; 123 and 61; 123 and 62; 123 and 63; 123 and 64; 124 and 1; 124 and 2; 124 and 3;

124 and 4; 124 and 5; 124 and 6; 124 and 7; 124 and 8; 124 and 9; 124 and 10; 124 and 11; 124 and 12; 124 and 13; 124 and 14; 124 and 15; 124 and 16; 124 and 17; 124 and 18; 124 and 19; 124 and 20; 124 and 21; 124 and 22; 124 and 23; 124 and 24; 124 and 25; 124 and 26; 124 and 27; 124 and 28; 124 and 29; 124 and 30; 124 and 31; 124 and 32; 124 and 33; 124 and 34; 124 and 35; 124 and 36; 124 and 37; 124 and 38; 124 and 39; 124 and 40; 124 and 41; 124 and 42; 124 and 43; 124 and 44; 124 and 45; 124 and 46; 124 and 47; 124 and 48; 124 and 49; 124 and 50; 124 and 51; 124 and 52; 124 and 53; 124 and 54; 124 and 55; 124 and 56; 124 and 57; 124 and 58; 124 and 59; 124 and 60; 124 and 61; 124 and 62; 124 and 63; 124 and 64; 125 and 1; 125 and 2; 125 and 3; 125 and 4; 125 and 5; 125 and 6; 125 and 7; 125 and 8; 125 and 9; 125 and 10; 125 and 11; 125 and 12; 125 and 13; 125 and 14; 125 and 15; 125 and 16; 125 and 17; 125 and 18; 125 and 19; 125 and 20; 125 and 21; 125 and 22; 125 and 23; 125 and 24; 125 and 25; 125 and 26; 125 and 27; 125 and 28; 125 and 29; 125 and 30; 125 and 31; 125 and 32; 125 and 33; 125 and 34; 125 and 35; 125 and 36; 125 and 37; 125 and 38; 125 and 39; 125 and 40; 125 and 41; 125 and 42; 125 and 43; 125 and 44; 125 and 45; 125 and 46; 125 and 47; 125 and 48; 125 and 49; 125 and 50; 125 and 51; 125 and 52; 125 and 53; 125 and 54; 125 and 55; 125 and 56; 125 and 57; 125 and 58; 125 and 59; 125 and 60; 125 and 61; 125 and 62; 125 and 63;

125 and 64; 126 and 1; 126 and 2; 126 and 3; 126 and 4; 126 and 5; 126 and 6; 126 and 7; 126 and 8; 126 and 9; 126 and 10; 126 and 11; 126 and 12; 126 and 13; 126 and 14; 126 and 15; 126 and 16; 126 and 17; 126 and 18; 126 and 19; 126 and 20; 126 and 21; 126 and 22; 126 and 23;

126 and 24; 126 and 25; 126 and 26; 126 and 27; 126 and 28; 126 and 29; 126 and 30; 126 and 31; 126 and 32; 126 and 33; 126 and 34; 126 and 35; 126 and 36; 126 and 37; 126 and 38; 126 and 39; 126 and 40; 126 and 41; 126 and 42; 126 and 43; 126 and 44; 126 and 45; 126 and 46;

126 and 47; 126 and 48; 126 and 49; 126 and 50; 126 and 51; 126 and 52; 126 and 53; 126 and 54; 126 and 55; 126 and 56; 126 and 57; 126 and 58; 126 and 59; 126 and 60; 126 and 61; 126 and 62; 126 and 63; 126 and 64; 127 and 1; 127 and 2; 127 and 3; 127 and 4; 127 and 5; 127 and 6; 127 and 7; 127 and 8; 127 and 9; 127 and 10; 127 and 11; 127 and 12; 127 and 13; 127 and 14; 127 and 15; 127 and 16; 127 and 17; 127 and 18; 127 and 19; 127 and 20; 127 and 21;

127 and 22; 127 and 23; 127 and 24; 127 and 25; 127 and 26; 127 and 27; 127 and 28; 127 and 29; 127 and 30; 127 and 31; 127 and 32; 127 and 33; 127 and 34; 127 and 35; 127 and 36; 127 and 37; 127 and 38; 127 and 39; 127 and 40; 127 and 41; 127 and 42; 127 and 43; 127 and 44;

127 and 45; 127 and 46; 127 and 47; 127 and 48; 127 and 49; 127 and 50; 127 and 51; 127 and 52; 127 and 53; 127 and 54; 127 and 55; 127 and 56; 127 and 57; 127 and 58; 127 and 59; 127 and 60; 127 and 61; 127 and 62; 127 and 63; 127 and 64; 128 and 1; 128 and 2; 128 and 3; 128 and 4; 128 and 5; 128 and 6; 128 and 7; 128 and 8; 128 and 9; 128 and 10; 128 and 11; 128 and 12; 128 and 13; 128 and 14; 128 and 15; 128 and 16; 128 and 17; 128 and 18; 128 and 19; 128 and 20; 128 and 21; 128 and 22; 128 and 23; 128 and 24; 128 and 25; 128 and 26; 128 and 27;

128 and 28; 128 and 29; 128 and 30; 128 and 31; 128 and 32; 128 and 33; 128 and 34; 128 and 35; 128 and 36; 128 and 37; 128 and 38; 128 and 39; 128 and 40; 128 and 41; 128 and 42; 128 and 43; 128 and 44; 128 and 45; 128 and 46; 128 and 47; 128 and 48; 128 and 49; 128 and 50; 128 and 51; 128 and 52; 128 and 53; 128 and 54; 128 and 55; 128 and 56; 128 and 57; 128 and 58; 128 and 59; 128 and 60; 128 and 61; 128 and 62; 128 and 63; 128 and 64; 129 and 1; 129 and 2; 129 and 3; 129 and 4; 129 and 5; 129 and 6; 129 and 7; 129 and 8; 129 and 9; 129 and 10; 129 and 11; 129 and 12; 129 and 13; 129 and 14; 129 and 15; 129 and 16; 129 and 17; 129 and 18; 129 and 19; 129 and 20; 129 and 21; 129 and 22; 129 and 23; 129 and 24; 129 and 25; 129 and 26; 129 and 27; 129 and 28; 129 and 29; 129 and 30; 129 and 31; 129 and 32; 129 and 33; 129 and 34; 129 and 35; 129 and 36; 129 and 37; 129 and 38; 129 and 39; 129 and 40; 129 and 41; 129 and 42; 129 and 43; 129 and 44; 129 and 45; 129 and 46; 129 and 47; 129 and 48;

129 and 49; 129 and 50; 129 and 51; 129 and 52; 129 and 53; 129 and 54; 129 and 55; 129 and 56; 129 and 57; 129 and 58; 129 and 59; 129 and 60; 129 and 61; 129 and 62; 129 and 63; 129 and 64; 130 and 1; 130 and 2; 130 and 3; 130 and 4; 130 and 5; 130 and 6; 130 and 7; 130 and 8; 130 and 9; 130 and 10; 130 and 11; 130 and 12; 130 and 13; 130 and 14; 130 and 15; 130 and 16; 130 and 17; 130 and 18; 130 and 19; 130 and 20; 130 and 21; 130 and 22; 130 and 23; 130 and 24; 130 and 25; 130 and 26; 130 and 27; 130 and 28; 130 and 29; 130 and 30; 130 and 31;

130 and 32; 130 and 33; 130 and 34; 130 and 35; 130 and 36; 130 and 37; 130 and 38; 130 and 39; 130 and 40; 130 and 41; 130 and 42; 130 and 43; 130 and 44; 130 and 45; 130 and 46; 130 and 47; 130 and 48; 130 and 49; 130 and 50; 130 and 51; 130 and 52; 130 and 53; 130 and 54;

130 and 55; 130 and 56; 130 and 57; 130 and 58; 130 and 59; 130 and 60; 130 and 61; 130 and 62; 130 and 63; 130 and 64; 131 and 1; 131 and 2; 131 and 3; 131 and 4; 131 and 5; 131 and 6;

131 and 7; 131 and 8; 131 and 9; 131 and 10; 131 and 11; 131 and 12; 131 and 13; 131 and 14; 131 and 15; 131 and 16; 131 and 17; 131 and 18; 131 and 19; 131 and 20; 131 and 21; 131 and 22; 131 and 23; 131 and 24; 131 and 25; 131 and 26; 131 and 27; 131 and 28; 131 and 29; 131 and 30; 131 and 31; 131 and 32; 131 and 33; 131 and 34; 131 and 35; 131 and 36; 131 and 37; 131 and 38; 131 and 39; 131 and 40; 131 and 41; 131 and 42; 131 and 43; 131 and 44; 131 and 45; 131 and 46; 131 and 47; 131 and 48; 131 and 49; 131 and 50; 131 and 51; 131 and 52; 131 and 53; 131 and 54; 131 and 55; 131 and 56; 131 and 57; 131 and 58; 131 and 59; 131 and 60;

131 and 61; 131 and 62; 131 and 63; 131 and 64; 132 and 1; 132 and 2; 132 and 3; 132 and 4;

132 and 5; 132 and 6; 132 and 7; 132 and 8; 132 and 9; 132 and 10; 132 and 11; 132 and 12; 132 and 13; 132 and 14; 132 and 15; 132 and 16; 132 and 17; 132 and 18; 132 and 19; 132 and 20; 132 and 21; 132 and 22; 132 and 23; 132 and 24; 132 and 25; 132 and 26; 132 and 27; 132 and 28; 132 and 29; 132 and 30; 132 and 31; 132 and 32; 132 and 33; 132 and 34; 132 and 35; 132 and 36; 132 and 37; 132 and 38; 132 and 39; 132 and 40; 132 and 41; 132 and 42; 132 and 43; 132 and 44; 132 and 45; 132 and 46; 132 and 47; 132 and 48; 132 and 49; 132 and 50; 132 and 51; 132 and 52; 132 and 53; 132 and 54; 132 and 55; 132 and 56; 132 and 57; 132 and 58;

132 and 59; 132 and 60; 132 and 61; 132 and 62; 132 and 63; 132 and 64; 133 and 1; 133 and 2;

133 and 3; 133 and 4; 133 and 5; 133 and 6; 133 and 7; 133 and 8; 133 and 9; 133 and 10; 133 and 11; 133 and 12; 133 and 13; 133 and 14; 133 and 15; 133 and 16; 133 and 17; 133 and 18;

133 and 19; 133 and 20; 133 and 21; 133 and 22; 133 and 23; 133 and 24; 133 and 25; 133 and

26; 133 and 27; 133 and 28; 133 and 29; 133 and 30; 133 and 31; 133 and 32; 133 and 33; 133 and 34; 133 and 35; 133 and 36; 133 and 37; 133 and 38; 133 and 39; 133 and 40; 133 and 41;

133 and 42; 133 and 43; 133 and 44; 133 and 45; 133 and 46; 133 and 47; 133 and 48; 133 and 49; 133 and 50; 133 and 51; 133 and 52; 133 and 53; 133 and 54; 133 and 55; 133 and 56; 133 and 57; 133 and 58; 133 and 59; 133 and 60; 133 and 61; 133 and 62; 133 and 63; 133 and 64; 134 and 1; 134 and 2; 134 and 3; 134 and 4; 134 and 5; 134 and 6; 134 and 7; 134 and 8; 134 and 9; 134 and 10; 134 and 11; 134 and 12; 134 and 13; 134 and 14; 134 and 15; 134 and 16; 134 and 17; 134 and 18; 134 and 19; 134 and 20; 134 and 21; 134 and 22; 134 and 23; 134 and 24; 134 and 25; 134 and 26; 134 and 27; 134 and 28; 134 and 29; 134 and 30; 134 and 31; 134 and 32; 134 and 33; 134 and 34; 134 and 35; 134 and 36; 134 and 37; 134 and 38; 134 and 39; 134 and 40; 134 and 41; 134 and 42; 134 and 43; 134 and 44; 134 and 45; 134 and 46; 134 and 47; 134 and 48; 134 and 49; 134 and 50; 134 and 51; 134 and 52; 134 and 53; 134 and 54; 134 and 55; 134 and 56; 134 and 57; 134 and 58; 134 and 59; 134 and 60; 134 and 61; 134 and 62;

134 and 63; 134 and 64; 135 and 1; 135 and 2; 135 and 3; 135 and 4; 135 and 5; 135 and 6; 135 and 7; 135 and 8; 135 and 9; 135 and 10; 135 and 11; 135 and 12; 135 and 13; 135 and 14; 135 and 15; 135 and 16; 135 and 17; 135 and 18; 135 and 19; 135 and 20; 135 and 21; 135 and 22;

135 and 23; 135 and 24; 135 and 25; 135 and 26; 135 and 27; 135 and 28; 135 and 29; 135 and 30; 135 and 31; 135 and 32; 135 and 33; 135 and 34; 135 and 35; 135 and 36; 135 and 37; 135 and 38; 135 and 39; 135 and 40; 135 and 41; 135 and 42; 135 and 43; 135 and 44; 135 and 45;

135 and 46; 135 and 47; 135 and 48; 135 and 49; 135 and 50; 135 and 51; 135 and 52; 135 and 53; 135 and 54; 135 and 55; 135 and 56; 135 and 57; 135 and 58; 135 and 59; 135 and 60; 135 and 61; 135 and 62; 135 and 63; 135 and 64; 136 and 1; 136 and 2; 136 and 3; 136 and 4; 136 and 5; 136 and 6; 136 and 7; 136 and 8; 136 and 9; 136 and 10; 136 and 11; 136 and 12; 136 and 13; 136 and 14; 136 and 15; 136 and 16; 136 and 17; 136 and 18; 136 and 19; 136 and 20;

136 and 21; 136 and 22; 136 and 23; 136 and 24; 136 and 25; 136 and 26; 136 and 27; 136 and 28; 136 and 29; 136 and 30; 136 and 31; 136 and 32; 136 and 33; 136 and 34; 136 and 35; 136 and 36; 136 and 37; 136 and 38; 136 and 39; 136 and 40; 136 and 41; 136 and 42; 136 and 43;

136 and 44; 136 and 45; 136 and 46; 136 and 47; 136 and 48; 136 and 49; 136 and 50; 136 and 51; 136 and 52; 136 and 53; 136 and 54; 136 and 55; 136 and 56; 136 and 57; 136 and 58; 136 and 59; 136 and 60; 136 and 61; 136 and 62; 136 and 63; 136 and 64; 137 and 1; 137 and 2; 137 and 3; 137 and 4; 137 and 5; 137 and 6; 137 and 7; 137 and 8; 137 and 9; 137 and 10; 137 and 11; 137 and 12; 137 and 13; 137 and 14; 137 and 15; 137 and 16; 137 and 17; 137 and 18; 137 and 19; 137 and 20; 137 and 21; 137 and 22; 137 and 23; 137 and 24; 137 and 25; 137 and 26;

137 and 27; 137 and 28; 137 and 29; 137 and 30; 137 and 31; 137 and 32; 137 and 33; 137 and 34; 137 and 35; 137 and 36; 137 and 37; 137 and 38; 137 and 39; 137 and 40; 137 and 41; 137 and 42; 137 and 43; 137 and 44; 137 and 45; 137 and 46; 137 and 47; 137 and 48; 137 and 49;

137 and 50; 137 and 51; 137 and 52; 137 and 53; 137 and 54; 137 and 55; 137 and 56; 137 and 57; 137 and 58; 137 and 59; 137 and 60; 137 and 61; 137 and 62; 137 and 63; 137 and 64; 138 and 1; 138 and 2; 138 and 3; 138 and 4; 138 and 5; 138 and 6; 138 and 7; 138 and 8; 138 and 9;

138 and 10; 138 and 11; 138 and 12; 138 and 13; 138 and 14; 138 and 15; 138 and 16; 138 and 17; 138 and 18; 138 and 19; 138 and 20; 138 and 21; 138 and 22; 138 and 23; 138 and 24; 138 and 25; 138 and 26; 138 and 27; 138 and 28; 138 and 29; 138 and 30; 138 and 31; 138 and 32; 138 and 33; 138 and 34; 138 and 35; 138 and 36; 138 and 37; 138 and 38; 138 and 39; 138 and 40; 138 and 41; 138 and 42; 138 and 43; 138 and 44; 138 and 45; 138 and 46; 138 and 47; 138 and 48; 138 and 49; 138 and 50; 138 and 51; 138 and 52; 138 and 53; 138 and 54; 138 and 55;

138 and 56; 138 and 57; 138 and 58; 138 and 59; 138 and 60; 138 and 61; 138 and 62; 138 and 63; 138 and 64; 139 and 1; 139 and 2; 139 and 3; 139 and 4; 139 and 5; 139 and 6; 139 and 7;

139 and 8; 139 and 9; 139 and 10; 139 and 11; 139 and 12; 139 and 13; 139 and 14; 139 and 15; 139 and 16; 139 and 17; 139 and 18; 139 and 19; 139 and 20; 139 and 21; 139 and 22; 139 and 23; 139 and 24; 139 and 25; 139 and 26; 139 and 27; 139 and 28; 139 and 29; 139 and 30; 139 and 31; 139 and 32; 139 and 33; 139 and 34; 139 and 35; 139 and 36; 139 and 37; 139 and 38; 139 and 39; 139 and 40; 139 and 41; 139 and 42; 139 and 43; 139 and 44; 139 and 45; 139 and 46; 139 and 47; 139 and 48; 139 and 49; 139 and 50; 139 and 51; 139 and 52; 139 and 53; 139 and 54; 139 and 55; 139 and 56; 139 and 57; 139 and 58; 139 and 59; 139 and 60; 139 and 61;

139 and 62; 139 and 63; 139 and 64; 140 and 1; 140 and 2; 140 and 3; 140 and 4; 140 and 5;

140 and 6; 140 and 7; 140 and 8; 140 and 9; 140 and 10; 140 and 11; 140 and 12; 140 and 13; 140 and 14; 140 and 15; 140 and 16; 140 and 17; 140 and 18; 140 and 19; 140 and 20; 140 and 21; 140 and 22; 140 and 23; 140 and 24; 140 and 25; 140 and 26; 140 and 27; 140 and 28; 140 and 29; 140 and 30; 140 and 31; 140 and 32; 140 and 33; 140 and 34; 140 and 35; 140 and 36; 140 and 37; 140 and 38; 140 and 39; 140 and 40; 140 and 41; 140 and 42; 140 and 43; 140 and 44; 140 and 45; 140 and 46; 140 and 47; 140 and 48; 140 and 49; 140 and 50; 140 and 51; 140 and 52; 140 and 53; 140 and 54; 140 and 55; 140 and 56; 140 and 57; 140 and 58; 140 and 59;

140 and 60; 140 and 61; 140 and 62; 140 and 63; 140 and 64; 141 and 1; 141 and 2; 141 and 3;

141 and 4; 141 and 5; 141 and 6; 141 and 7; 141 and 8; 141 and 9; 141 and 10; 141 and 11; 141 and 12; 141 and 13; 141 and 14; 141 and 15; 141 and 16; 141 and 17; 141 and 18; 141 and 19; 141 and 20; 141 and 21; 141 and 22; 141 and 23; 141 and 24; 141 and 25; 141 and 26; 141 and 27; 141 and 28; 141 and 29; 141 and 30; 141 and 31; 141 and 32; 141 and 33; 141 and 34; 141 and 35; 141 and 36; 141 and 37; 141 and 38; 141 and 39; 141 and 40; 141 and 41; 141 and 42;

141 and 43; 141 and 44; 141 and 45; 141 and 46; 141 and 47; 141 and 48; 141 and 49; 141 and 50; 141 and 51; 141 and 52; 141 and 53; 141 and 54; 141 and 55; 141 and 56; 141 and 57; 141 and 58; 141 and 59; 141 and 60; 141 and 61; 141 and 62; 141 and 63; 141 and 64; 142 and 1;

142 and 2; 142 and 3; 142 and 4; 142 and 5; 142 and 6; 142 and 7; 142 and 8; 142 and 9; 142 and 10; 142 and 11; 142 and 12; 142 and 13; 142 and 14; 142 and 15; 142 and 16; 142 and 17; 142 and 18; 142 and 19; 142 and 20; 142 and 21; 142 and 22; 142 and 23; 142 and 24; 142 and 25; 142 and 26; 142 and 27; 142 and 28; 142 and 29; 142 and 30; 142 and 31; 142 and 32; 142 and 33; 142 and 34; 142 and 35; 142 and 36; 142 and 37; 142 and 38; 142 and 39; 142 and 40; 142 and 41; 142 and 42; 142 and 43; 142 and 44; 142 and 45; 142 and 46; 142 and 47; 142 and 48; 142 and 49; 142 and 50; 142 and 51; 142 and 52; 142 and 53; 142 and 54; 142 and 55; 142 and 56; 142 and 57; 142 and 58; 142 and 59; 142 and 60; 142 and 61; 142 and 62; 142 and 63;

142 and 64; 143 and 1; 143 and 2; 143 and 3; 143 and 4; 143 and 5; 143 and 6; 143 and 7; 143 and 8; 143 and 9; 143 and 10; 143 and 11; 143 and 12; 143 and 13; 143 and 14; 143 and 15; 143 and 16; 143 and 17; 143 and 18; 143 and 19; 143 and 20; 143 and 21; 143 and 22; 143 and 23;

143 and 24; 143 and 25; 143 and 26; 143 and 27; 143 and 28; 143 and 29; 143 and 30; 143 and 31; 143 and 32; 143 and 33; 143 and 34; 143 and 35; 143 and 36; 143 and 37; 143 and 38; 143 and 39; 143 and 40; 143 and 41; 143 and 42; 143 and 43; 143 and 44; 143 and 45; 143 and 46; 143 and 47; 143 and 48; 143 and 49; 143 and 50; 143 and 51; 143 and 52; 143 and 53; 143 and 54; 143 and 55; 143 and 56; 143 and 57; 143 and 58; 143 and 59; 143 and 60; 143 and 61; 143 and 62; 143 and 63; or 143 and 64; or b. SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28; 113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48; 113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15;

113 and 16; 113 and 17; 113 and 57; 113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and

59; 113 and 23; 113 and 24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and

48; 115 and 13; 115 and 53; 115 and 54; 115 and 55; 115 and 56; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18; 115 and 58; 115 and 19; 115 and 20; 115 and 59;

115 and 23; 115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and

26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42.

[00115] In some embodiments, a composition is provided comprising: one or more nucleic acid molecules encoding a SluCas9 and at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first guide sequence and a second guide sequence selected from any of the pairs of guide sequences shown in Table 1D.

[00116] In some embodiments, the disclosure provides for one or more nucleic acid molecules encoding a pair of guide RNAs comprising a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA target intron 52 and intron 53 of the DMD gene. In some embodiments, the guide RNAs are for use with a SluCas9 endonuclease. In some embodiments, the one or more nucleic acid molecules also encode for a SluCas9 endonuclease. In some embodiments, the one or more nucleic acid molecules are in a composition further comprising a SluCas9 endonuclease. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 6. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 28. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 35. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 36. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 37. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 38. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 39. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 40. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 41. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 42. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 9. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 44. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 10. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 45. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 46. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 11. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 47. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 12. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 48. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 13. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 53. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 54. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 55. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 56. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 14. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 15. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 16. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 17. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 57. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 18. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 58. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 19. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 20. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 59. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 23. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 24. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 60. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 62. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 63. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 64. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 25. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 113 and the second guide RNA comprises the sequence of SEQ ID NO: 26. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 6. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 28. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 35. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 36. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 37. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 38. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 39. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 40. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 41. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 42. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 9. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 44. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 10. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 45. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 46. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 11. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 47. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 12. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 48. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 13. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 53. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 54. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 55. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 56. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 14. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 15. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 16. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 17. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 57. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 18. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 58. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 19. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 20. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 59. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 23. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 24. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 60. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 62. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 63. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 64. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 25. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 26. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

116 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

120 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 6. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

121 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 6. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 122 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 122 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 122 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 122 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 122 and the second guide RNA comprises the sequence of SEQ ID NO: 6. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 122 and the second guide RNA comprises the sequence of SEQ ID NO: 28. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 6. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 28. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 35. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 36. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 37. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 38. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

92 and the second guide RNA comprises the sequence of SEQ ID NO: 39. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 40. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 41. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 92 and the second guide RNA comprises the sequence of SEQ ID NO: 42. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 1. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

93 and the second guide RNA comprises the sequence of SEQ ID NO: 2. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 3. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 5. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 6. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 28. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 35. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 36. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 37. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 38. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 39. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 40. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 41. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 93 and the second guide RNA comprises the sequence of SEQ ID NO: 42. In some embodiments the guide RNAs each comprise a scaffold sequence comprising the sequence of SEQ ID NO: 901.

[00117] In some embodiments, a composition is provided comprising or consisting of a single nucleic acid molecule comprising: (i) a nucleic acid encoding SluCas9 and at least one, at least two, or at least three guide RNAs; or (ii) a nucleic acid encoding SluCas9 and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or (iii) a nucleic acid encoding SluCas9 and 1, 2, or 3 guide RNAs. In such embodiments, each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A or 1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide sequence selected from the pairs of guide sequences shown in Table 1D, and further optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence.

[00118] In some embodiments, a composition is provided comprising or consisting of two nucleic acid molecules comprising: i. a first nucleic acid encoding SluCas9; and a second nucleic acid that does not encode a SluCas9 and encodes any one of the following: 1. at least one, at least two, at least three, at least four, at least five, or at least six guide

RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; ii. a first nucleic acid encoding SluCas9 and

1. at least one, at least two, or at least three guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. 1, 2, or 3 guide RNAs; and a second nucleic acid that does not encode a SluCas9, optionally wherein the second nucleic acid comprises any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; iii. a first nucleic acid encoding SluCas9 and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SluCas9 and encodes from one to six guide RNAs; or iv. a first nucleic acid encoding SluCas9 and at least two guide RNAs, and a second nucleic acid that does not encode a SluCas9 and encodes at least one additional copy of each of the guide RNAs encoded in the first nucleic acid; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A or 1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from the pairs of guide sequences shown in Table 1D, and optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence.

B. Guide pairs targeting intron 52 and exon 53, or exon 53 and intron 53 of the DMD gene

[00119] In some embodiments, a composition is provided comprising at least one pair of guide RNAs, wherein the at least one pair of guide RNAs is capable of excising a fragment of exon 53 of the DMD gene, and wherein the fragment comprises a splice acceptor site, a first guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases upstream of the splice acceptor site, and a second guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases downstream of the splice acceptor site (Figure 4, middle). In particular embodiments, the first guide RNA targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases upstream of the splice acceptor site, and the second guide RNA targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases downstream of the splice acceptor site. In some embodiments, the fragment of the DMD gene comprises a splice acceptor site and further comprises a portion of exon 53 and an adjoining portion of intron 52 of the DMD gene. In particular embodiments, the first guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 1-200 of SEQ ID NO: 144, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 201-400 of SEQ ID NO: 144. In SEQ ID NO: 144 shown below, the splice acceptor site (nucleotides 175-202 of SEQ ID NO: 144) is underlined.

[00120] SEQ ID NO: 144:

TCAGGAAAATAATTTAACAGAAACTAATATTTGCCATCAAAAAAGCAAAGAATCCTGTT GTTCATCATCCTAGCCATAACACAATGAATAATTTTTTAAATAAGCAACATAAATGTGAG ATAACGTTTGGAAGTTACATTTAAAATGTCTCCTCCAGACTAGCATTTACTACTATATATT TATTTTTCCTTTTATTCTAGTTGAAAGAATTCAGAATCAGTGGGATGAAGTACAAGAACA CCTTCAGAACCGGAGGCAACAGTTGAATGAAATGTTAAAGGATTCAACACAATGGCTGG AAGCTAAGGAAGAAGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTCATGG AAGGAGGGTCCCTATACAGTAGATGCAATCCAAAAGAAAATC.

[00121] In some embodiments, a composition is provided comprising at least one pair of guide RNAs, wherein the at least one pair of guide RNAs is capable of excising a fragment of exon 53 of the DMD gene, and wherein the fragment comprises a splice donor site, a first guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases upstream of the splice donor site, and a second guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases downstream of the splice donor site (Figure 4, bottom). In particular embodiments, the first guide RNA targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases upstream of the splice donor site, and the second guide RNA targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases downstream of the splice donor site. In some embodiments, the fragment of the DMD gene comprises a splice donor site and further comprises a portion of intron 53 and an adjoining portion of exon 53 of the DMD gene. In particular embodiments, the first guide RNA targets a sequence within nucleotides 1-200 of SEQ ID NO: 145, and the second guide RNA targets a sequence within nucleobases 201-400 of SEQ ID NO: 145. In SEQ ID NO: 145 shown below, the splice donor site (nucleobases 198-207 of SEQ ID NO: 145) is underlined.

[00122] SEQ ID NO: 145:

AGAATCAGTGGGATGAAGTACAAGAACACCTTCAGAACCGGAGGCAACAGTTGAATGA AATGTTAAAGGATTCAACACAATGGCTGGAAGCTAAGGAAGAAGCTGAGCAGGTCTTAG GACAGGCCAGAGCCAAGCTTGAGTCATGGAAGGAGGGTCCCTATACAGTAGATGCAATC CAAAAGAAAATCACAGAAACCAAGGTTAGTATCAAAGATACCTTTTTAAAATAAAATAC TGGTTACATTTGATAAAATTATACCATAGATTGTAATTTAATGATGTTTAATGTAAAGTTA TTAACAGAAAATCACGTTAAAGCTGAAATGAACAGTAGACTTTGTATATTTATTTTCTTA GAGACAGAGTCTCACTGTCACCCAGGCTAAAGTGCAGTGGCACA.

[00123] Any of the above embodiments may further comprise an endonuclease or a nucleic acid encoding an endonuclease, optionally a SluCas9 or a nucleic acid encoding a SluCas9.

[00124] In some embodiments, a composition is provided comprising a pair of guide RNAs, wherein the pair of guide RNAs comprises or consists of any one of the pairs of guide sequences of any one of Table 1E. In some embodiments, a composition is provided comprising a pair of guide RNAs, wherein the pair of guide RNAs comprises or consists of any one of the pairs of guide sequences of any one of Table 1F.

[00125] In some embodiments, a composition is provided comprising a pair of guide RNAs or a nucleic acid encoding a pair of guide RNAs, wherein the first guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-64; and the second guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 65-87. In some embodiments, a composition is provided comprising a pair of guide RNAs or a nucleic acid encoding a pair of guide RNAs, wherein the first guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 65- 87; and the second guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 88-143. In some embodiments, a composition is provided comprising a pair of guide RNAs or a nucleic acid encoding a pair of guide RNAs, wherein the first guide RNA is selected from any one of SEQ ID NOs: 1-64; and the second guide RNA is selected from any one of SEQ ID NOs: 65-87. In some embodiments, a composition is provided comprising a pair of guide RNAs or a nucleic acid encoding a pair of guide RNAs, wherein the first guide RNA is selected from any one of SEQ ID NOs: 65-87; and the second guide RNA is selected from any one of SEQ ID NOs: 88-143. In some embodiments, the first guide RNA targets a sequence in intron 53 of a DMD gene and the second guide RNA targets a sequence in exon 53 of the DMD gene. In some embodiments, the first guide RNA targets a sequence in exon 53 of a DMD gene and the second guide RNA targets a sequence in intron 52 of the DMD gene. In some embodiments, the at least one pair of guide RNAs is capable of excising a fragment of the DMD gene, wherein the fragment comprises a splice donor site or a splice acceptor site, and optionally wherein the fragment is between 5 and 400 nucleotides in length. In particular embodiments, the excised fragment of the DMD gene does not comprise an entire exon of the DMD gene. In some embodiments, the pair of guide RNAs is capable of inducing skipping of at least a portion (such as all) of exon 53 of the DMD gene.

[00126] In some embodiments, a composition is provided comprising at least one pair of guide RNAs disclosed herein, once expressed in vitro or in vivo in combination with a SluCas9 endonuclease, the at least one pair of guide RNAs (i) excises exon 53 of a DMD gene, (ii) excises a fragment of the DMD gene comprising a portion of intron 53 and an adjoining portion of exon 53, wherein the excised fragment of the DMD gene comprises a splice donor site, or (iii) excises a fragment of the DMD gene comprising a portion of exon 53 and an adjoining portion of intron 52, wherein the excised fragment of the DMD gene comprises a splice acceptor site. In some such embodiments, the size of the excised fragment of the DMD gene is between 5 and 400 nucleotides in length, such as between 5 and 375, 5 and 350, 5 and 325, 5 and 300, 5 and 275, 5 and 250, 5 and 225, 5 and 200, 5 and 175, 5 and 150, 5 and 125, 5 and 100, 20 and 400, 20 and 375, 20 and 350, 20 and 325, 20 and 300, 20 and 275, 20 and 250, 20 and 225, 20 and 200, 20 and 175, 20 and 150, 20 and 125, 20 and 100, 50 and 400, 50 and 375, 50 and 350, 50 and 325, 50 and 300, 50 and 275, 50 and 250, 50 and 225, 50 and 200, 50 and 175, 50 and 150, 50 and 125, or 50 and 100 nucleotides in length.

[00127] In some embodiments, a composition is provided comprising at least one pair of guide RNAs or a nucleic acid encoding at least one pair of guide RNAs, wherein the first and the second guide RNAs of the at least one pair of guide RNAs each comprise a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a first and a second guide sequence selected from any one of the following pairs of first and second guide sequences: a. SEQ ID NOs: 1 and 65; 1 and 66; 1 and 67; 1 and 68; 1 and 69; 1 and 70; 1 and 71; 1 and 72; 1 and 73; 1 and 74; 1 and 75; 1 and 76; 1 and 77; 1 and 78; 1 and 79; 1 and 80; 1 and 81; 1 and 82; 1 and 83; 1 and 84; 1 and 85; 1 and 86; 1 and 87; 2 and 65; 2 and 66; 2 and 67; 2 and 68; 2 and 69; 2 and 70; 2 and 71; 2 and 72; 2 and 73; 2 and 74; 2 and 75; 2 and 76; 2 and 77; 2 and 78; 2 and 79; 2 and 80; 2 and 81; 2 and 82; 2 and 83; 2 and 84; 2 and 85; 2 and 86; 2 and 87; 3 and 65; 3 and 66; 3 and 67; 3 and 68; 3 and 69; 3 and 70; 3 and 71; 3 and 72; 3 and 73; 3 and 74; 3 and 75; 3 and 76; 3 and 77; 3 and 78; 3 and 79; 3 and 80; 3 and 81; 3 and 82; 3 and 83; 3 and 84; 3 and 85; 3 and 86; 3 and 87; 4 and 65; 4 and 66; 4 and 67; 4 and 68; 4 and 69; 4 and 70; 4 and 71; 4 and 72; 4 and 73; 4 and 74; 4 and 75; 4 and 76; 4 and 77; 4 and 78; 4 and 79; 4 and 80; 4 and 81; 4 and 82; 4 and 83; 4 and 84; 4 and 85; 4 and 86; 4 and 87; 5 and 65; 5 and 66; 5 and 67; 5 and 68; 5 and 69; 5 and 70; 5 and 71; 5 and 72; 5 and 73; 5 and 74; 5 and 75; 5 and 76; 5 and 77; 5 and 78; 5 and 79; 5 and 80; 5 and 81; 5 and 82; 5 and 83; 5 and 84; 5 and 85; 5 and 86; 5 and 87; 6 and 65; 6 and 66; 6 and 67; 6 and 68; 6 and 69; 6 and 70; 6 and 71; 6 and 72; 6 and 73; 6 and 74; 6 and 75; 6 and 76; 6 and 77; 6 and 78; 6 and 79; 6 and 80; 6 and 81; 6 and 82; 6 and 83; 6 and 84; 6 and 85; 6 and 86; 6 and 87; 7 and 65; 7 and 66; 7 and 67; 7 and 68; 7 and 69; 7 and 70; 7 and 71; 7 and 72; 7 and 73; 7 and 74; 7 and 75; 7 and 76; 7 and 77; 7 and 78; 7 and 79; 7 and 80; 7 and 81; 7 and 82; 7 and 83; 7 and 84; 7 and 85; 7 and 86; 7 and 87; 8 and 65; 8 and 66; 8 and 67; 8 and 68; 8 and 69; 8 and 70; 8 and 71; 8 and 72; 8 and 73; 8 and 74; 8 and 75; 8 and 76; 8 and 77; 8 and 78; 8 and 79; 8 and 80; 8 and 81; 8 and 82; 8 and 83; 8 and 84; 8 and 85; 8 and 86; 8 and 87; 9 and 65; 9 and 66; 9 and 67; 9 and 68; 9 and 69; 9 and 70; 9 and 71; 9 and 72; 9 and 73; 9 and 74; 9 and 75; 9 and 76; 9 and 77; 9 and 78; 9 and 79; 9 and 80; 9 and 81; 9 and 82; 9 and 83; 9 and 84; 9 and 85; 9 and 86; 9 and 87; 10 and 65; 10 and 66; 10 and 67; 10 and 68; 10 and 69; 10 and 70; 10 and 71; 10 and 72; 10 and 73;

10 and 74; 10 and 75; 10 and 76; 10 and 77; 10 and 78; 10 and 79; 10 and 80; 10 and 81; 10 and 82; 10 and 83; 10 and 84; 10 and 85; 10 and 86; 10 and 87; 11 and 65; 11 and 66; 11 and 67; 11 and 68; 11 and 69; 11 and 70; 11 and 71; 11 and 72; 11 and 73; 11 and 74; 11 and 75;

11 and 76; 11 and 77; 11 and 78; 11 and 79; 11 and 80; 11 and 81; 11 and 82; 11 and 83; 11 and 84; 11 and 85; 11 and 86; 11 and 87; 12 and 65; 12 and 66; 12 and 67; 12 and 68; 12 and 69; 12 and 70; 12 and 71; 12 and 72; 12 and 73; 12 and 74; 12 and 75; 12 and 76; 12 and 77;

12 and 78; 12 and 79; 12 and 80; 12 and 81; 12 and 82; 12 and 83; 12 and 84; 12 and 85; 12 and 86; 12 and 87; 13 and 65; 13 and 66; 13 and 67; 13 and 68; 13 and 69; 13 and 70; 13 and 71; 13 and 72; 13 and 73; 13 and 74; 13 and 75; 13 and 76; 13 and 77; 13 and 78; 13 and 79;

13 and 80; 13 and 81; 13 and 82; 13 and 83; 13 and 84; 13 and 85; 13 and 86; 13 and 87; 14 and 65; 14 and 66; 14 and 67; 14 and 68; 14 and 69; 14 and 70; 14 and 71; 14 and 72; 14 and 73; 14 and 74; 14 and 75; 14 and 76; 14 and 77; 14 and 78; 14 and 79; 14 and 80; 14 and 81;

14 and 82; 14 and 83; 14 and 84; 14 and 85; 14 and 86; 14 and 87; 15 and 65; 15 and 66; 15 and 67; 15 and 68; 15 and 69; 15 and 70; 15 and 71; 15 and 72; 15 and 73; 15 and 74; 15 and 75; 15 and 76; 15 and 77; 15 and 78; 15 and 79; 15 and 80; 15 and 81; 15 and 82; 15 and 83;

15 and 84; 15 and 85; 15 and 86; 15 and 87; 16 and 65; 16 and 66; 16 and 67; 16 and 68; 16 and 69; 16 and 70; 16 and 71; 16 and 72; 16 and 73; 16 and 74; 16 and 75; 16 and 76; 16 and 77; 16 and 78; 16 and 79; 16 and 80; 16 and 81; 16 and 82; 16 and 83; 16 and 84; 16 and 85;

16 and 86; 16 and 87; 17 and 65; 17 and 66; 17 and 67; 17 and 68; 17 and 69; 17 and 70; 17 and 71; 17 and 72; 17 and 73; 17 and 74; 17 and 75; 17 and 76; 17 and 77; 17 and 78; 17 and 79; 17 and 80; 17 and 81; 17 and 82; 17 and 83; 17 and 84; 17 and 85; 17 and 86; 17 and 87;

18 and 65; 18 and 66; 18 and 67; 18 and 68; 18 and 69; 18 and 70; 18 and 71; 18 and 72; 18 and 73; 18 and 74; 18 and 75; 18 and 76; 18 and 77; 18 and 78; 18 and 79; 18 and 80; 18 and 81; 18 and 82; 18 and 83; 18 and 84; 18 and 85; 18 and 86; 18 and 87; 19 and 65; 19 and 66;

19 and 67; 19 and 68; 19 and 69; 19 and 70; 19 and 71; 19 and 72; 19 and 73; 19 and 74; 19 and 75; 19 and 76; 19 and 77; 19 and 78; 19 and 79; 19 and 80; 19 and 81; 19 and 82; 19 and

83; 19 and 84; 19 and 85; 19 and 86; 19 and 87; 20 and 65; 20 and 66; 20 and 67; 20 and 68;

20 and 69; 20 and 70; 20 and 71; 20 and 72; 20 and 73; 20 and 74; 20 and 75; 20 and 76; 20 and 77; 20 and 78; 20 and 79; 20 and 80; 20 and 81; 20 and 82; 20 and 83; 20 and 84; 20 and 85; 20 and 86; 20 and 87; 21 and 65; 21 and 66; 21 and 67; 21 and 68; 21 and 69; 21 and 70;

21 and 71; 21 and 72; 21 and 73; 21 and 74; 21 and 75; 21 and 76; 21 and 77; 21 and 78; 21 and 79; 21 and 80; 21 and 81; 21 and 82; 21 and 83; 21 and 84; 21 and 85; 21 and 86; 21 and 87; 22 and 65; 22 and 66; 22 and 67; 22 and 68; 22 and 69; 22 and 70; 22 and 71; 22 and 72;

22 and 73; 22 and 74; 22 and 75; 22 and 76; 22 and 77; 22 and 78; 22 and 79; 22 and 80; 22 and 81; 22 and 82; 22 and 83; 22 and 84; 22 and 85; 22 and 86; 22 and 87; 23 and 65; 23 and 66; 23 and 67; 23 and 68; 23 and 69; 23 and 70; 23 and 71; 23 and 72; 23 and 73; 23 and 74;

23 and 75; 23 and 76; 23 and 77; 23 and 78; 23 and 79; 23 and 80; 23 and 81; 23 and 82; 23 and 83; 23 and 84; 23 and 85; 23 and 86; 23 and 87; 24 and 65; 24 and 66; 24 and 67; 24 and 68; 24 and 69; 24 and 70; 24 and 71; 24 and 72; 24 and 73; 24 and 74; 24 and 75; 24 and 76;

24 and 77; 24 and 78; 24 and 79; 24 and 80; 24 and 81; 24 and 82; 24 and 83; 24 and 84; 24 and 85; 24 and 86; 24 and 87; 25 and 65; 25 and 66; 25 and 67; 25 and 68; 25 and 69; 25 and 70; 25 and 71; 25 and 72; 25 and 73; 25 and 74; 25 and 75; 25 and 76; 25 and 77; 25 and 78;

25 and 79; 25 and 80; 25 and 81; 25 and 82; 25 and 83; 25 and 84; 25 and 85; 25 and 86; 25 and 87; 26 and 65; 26 and 66; 26 and 67; 26 and 68; 26 and 69; 26 and 70; 26 and 71; 26 and 72; 26 and 73; 26 and 74; 26 and 75; 26 and 76; 26 and 77; 26 and 78; 26 and 79; 26 and 80;

26 and 81; 26 and 82; 26 and 83; 26 and 84; 26 and 85; 26 and 86; 26 and 87; 27 and 65; 27 and 66; 27 and 67; 27 and 68; 27 and 69; 27 and 70; 27 and 71; 27 and 72; 27 and 73; 27 and 74; 27 and 75; 27 and 76; 27 and 77; 27 and 78; 27 and 79; 27 and 80; 27 and 81; 27 and 82;

27 and 83; 27 and 84; 27 and 85; 27 and 86; 27 and 87; 28 and 65; 28 and 66; 28 and 67; 28 and 68; 28 and 69; 28 and 70; 28 and 71; 28 and 72; 28 and 73; 28 and 74; 28 and 75; 28 and 76; 28 and 77; 28 and 78; 28 and 79; 28 and 80; 28 and 81; 28 and 82; 28 and 83; 28 and 84;

28 and 85; 28 and 86; 28 and 87; 29 and 65; 29 and 66; 29 and 67; 29 and 68; 29 and 69; 29 and 70; 29 and 71; 29 and 72; 29 and 73; 29 and 74; 29 and 75; 29 and 76; 29 and 77; 29 and 78; 29 and 79; 29 and 80; 29 and 81; 29 and 82; 29 and 83; 29 and 84; 29 and 85; 29 and 86;

29 and 87; 30 and 65; 30 and 66; 30 and 67; 30 and 68; 30 and 69; 30 and 70; 30 and 71; 30 and 72; 30 and 73; 30 and 74; 30 and 75; 30 and 76; 30 and 77; 30 and 78; 30 and 79; 30 and 80; 30 and 81; 30 and 82; 30 and 83; 30 and 84; 30 and 85; 30 and 86; 30 and 87; 31 and 65;

31 and 66; 31 and 67; 31 and 68; 31 and 69; 31 and 70; 31 and 71; 31 and 72; 31 and 73; 31 and 74; 31 and 75; 31 and 76; 31 and 77; 31 and 78; 31 and 79; 31 and 80; 31 and 81; 31 and 82; 31 and 83; 31 and 84; 31 and 85; 31 and 86; 31 and 87; 32 and 65; 32 and 66; 32 and 67;

32 and 68; 32 and 69; 32 and 70; 32 and 71; 32 and 72; 32 and 73; 32 and 74; 32 and 75; 32 and 76; 32 and 77; 32 and 78; 32 and 79; 32 and 80; 32 and 81; 32 and 82; 32 and 83; 32 and 84; 32 and 85; 32 and 86; 32 and 87; 33 and 65; 33 and 66; 33 and 67; 33 and 68; 33 and 69;

33 and 70; 33 and 71; 33 and 72; 33 and 73; 33 and 74; 33 and 75; 33 and 76; 33 and 77; 33 and 78; 33 and 79; 33 and 80; 33 and 81; 33 and 82; 33 and 83; 33 and 84; 33 and 85; 33 and 86; 33 and 87; 34 and 65; 34 and 66; 34 and 67; 34 and 68; 34 and 69; 34 and 70; 34 and 71;

34 and 72; 34 and 73; 34 and 74; 34 and 75; 34 and 76; 34 and 77; 34 and 78; 34 and 79; 34 and 80; 34 and 81; 34 and 82; 34 and 83; 34 and 84; 34 and 85; 34 and 86; 34 and 87; 35 and 65; 35 and 66; 35 and 67; 35 and 68; 35 and 69; 35 and 70; 35 and 71; 35 and 72; 35 and 73;

35 and 74; 35 and 75; 35 and 76; 35 and 77; 35 and 78; 35 and 79; 35 and 80; 35 and 81; 35 and 82; 35 and 83; 35 and 84; 35 and 85; 35 and 86; 35 and 87; 36 and 65; 36 and 66; 36 and 67; 36 and 68; 36 and 69; 36 and 70; 36 and 71; 36 and 72; 36 and 73; 36 and 74; 36 and 75;

36 and 76; 36 and 77; 36 and 78; 36 and 79; 36 and 80; 36 and 81; 36 and 82; 36 and 83; 36 and 84; 36 and 85; 36 and 86; 36 and 87; 37 and 65; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 37 and 70; 37 and 71; 37 and 72; 37 and 73; 37 and 74; 37 and 75; 37 and 76; 37 and 77;

37 and 78; 37 and 79; 37 and 80; 37 and 81; 37 and 82; 37 and 83; 37 and 84; 37 and 85; 37 and 86; 37 and 87; 38 and 65; 38 and 66; 38 and 67; 38 and 68; 38 and 69; 38 and 70; 38 and 71; 38 and 72; 38 and 73; 38 and 74; 38 and 75; 38 and 76; 38 and 77; 38 and 78; 38 and 79;

38 and 80; 38 and 81; 38 and 82; 38 and 83; 38 and 84; 38 and 85; 38 and 86; 38 and 87; 39 and 65; 39 and 66; 39 and 67; 39 and 68; 39 and 69; 39 and 70; 39 and 71; 39 and 72; 39 and 73; 39 and 74; 39 and 75; 39 and 76; 39 and 77; 39 and 78; 39 and 79; 39 and 80; 39 and 81;

39 and 82; 39 and 83; 39 and 84; 39 and 85; 39 and 86; 39 and 87; 40 and 65; 40 and 66; 40 and 67; 40 and 68; 40 and 69; 40 and 70; 40 and 71; 40 and 72; 40 and 73; 40 and 74; 40 and 75; 40 and 76; 40 and 77; 40 and 78; 40 and 79; 40 and 80; 40 and 81; 40 and 82; 40 and 83;

40 and 84; 40 and 85; 40 and 86; 40 and 87; 41 and 65; 41 and 66; 41 and 67; 41 and 68; 41 and 69; 41 and 70; 41 and 71; 41 and 72; 41 and 73; 41 and 74; 41 and 75; 41 and 76; 41 and 77; 41 and 78; 41 and 79; 41 and 80; 41 and 81; 41 and 82; 41 and 83; 41 and 84; 41 and 85;

41 and 86; 41 and 87; 42 and 65; 42 and 66; 42 and 67; 42 and 68; 42 and 69; 42 and 70; 42 and 71; 42 and 72; 42 and 73; 42 and 74; 42 and 75; 42 and 76; 42 and 77; 42 and 78; 42 and 79; 42 and 80; 42 and 81; 42 and 82; 42 and 83; 42 and 84; 42 and 85; 42 and 86; 42 and 87;

43 and 65; 43 and 66; 43 and 67; 43 and 68; 43 and 69; 43 and 70; 43 and 71; 43 and 72; 43 and 73; 43 and 74; 43 and 75; 43 and 76; 43 and 77; 43 and 78; 43 and 79; 43 and 80; 43 and 81; 43 and 82; 43 and 83; 43 and 84; 43 and 85; 43 and 86; 43 and 87; 44 and 65; 44 and 66;

44 and 67; 44 and 68; 44 and 69; 44 and 70; 44 and 71; 44 and 72; 44 and 73; 44 and 74; 44 and 75; 44 and 76; 44 and 77; 44 and 78; 44 and 79; 44 and 80; 44 and 81; 44 and 82; 44 and 83; 44 and 84; 44 and 85; 44 and 86; 44 and 87; 45 and 65; 45 and 66; 45 and 67; 45 and 68;

45 and 69; 45 and 70; 45 and 71; 45 and 72; 45 and 73; 45 and 74; 45 and 75; 45 and 76; 45 and 77; 45 and 78; 45 and 79; 45 and 80; 45 and 81; 45 and 82; 45 and 83; 45 and 84; 45 and 85; 45 and 86; 45 and 87; 46 and 65; 46 and 66; 46 and 67; 46 and 68; 46 and 69; 46 and 70;

46 and 71; 46 and 72; 46 and 73; 46 and 74; 46 and 75; 46 and 76; 46 and 77; 46 and 78; 46 and 79; 46 and 80; 46 and 81; 46 and 82; 46 and 83; 46 and 84; 46 and 85; 46 and 86; 46 and 87; 47 and 65; 47 and 66; 47 and 67; 47 and 68; 47 and 69; 47 and 70; 47 and 71; 47 and 72; 47 and 73; 47 and 74; 47 and 75; 47 and 76; 47 and 77; 47 and 78; 47 and 79; 47 and 80; 47 and 81; 47 and 82; 47 and 83; 47 and 84; 47 and 85; 47 and 86; 47 and 87; 48 and 65; 48 and 66; 48 and 67; 48 and 68; 48 and 69; 48 and 70; 48 and 71; 48 and 72; 48 and 73; 48 and 74;

48 and 75; 48 and 76; 48 and 77; 48 and 78; 48 and 79; 48 and 80; 48 and 81; 48 and 82; 48 and 83; 48 and 84; 48 and 85; 48 and 86; 48 and 87; 49 and 65; 49 and 66; 49 and 67; 49 and 68; 49 and 69; 49 and 70; 49 and 71; 49 and 72; 49 and 73; 49 and 74; 49 and 75; 49 and 76;

49 and 77; 49 and 78; 49 and 79; 49 and 80; 49 and 81; 49 and 82; 49 and 83; 49 and 84; 49 and 85; 49 and 86; 49 and 87; 50 and 65; 50 and 66; 50 and 67; 50 and 68; 50 and 69; 50 and 70; 50 and 71; 50 and 72; 50 and 73; 50 and 74; 50 and 75; 50 and 76; 50 and 77; 50 and 78;

50 and 79; 50 and 80; 50 and 81; 50 and 82; 50 and 83; 50 and 84; 50 and 85; 50 and 86; 50 and 87; 51 and 65; 51 and 66; 51 and 67; 51 and 68; 51 and 69; 51 and 70; 51 and 71; 51 and 72; 51 and 73; 51 and 74; 51 and 75; 51 and 76; 51 and 77; 51 and 78; 51 and 79; 51 and 80;

51 and 81; 51 and 82; 51 and 83; 51 and 84; 51 and 85; 51 and 86; 51 and 87; 52 and 65; 52 and 66; 52 and 67; 52 and 68; 52 and 69; 52 and 70; 52 and 71; 52 and 72; 52 and 73; 52 and 74; 52 and 75; 52 and 76; 52 and 77; 52 and 78; 52 and 79; 52 and 80; 52 and 81; 52 and 82;

52 and 83; 52 and 84; 52 and 85; 52 and 86; 52 and 87; 53 and 65; 53 and 66; 53 and 67; 53 and 68; 53 and 69; 53 and 70; 53 and 71; 53 and 72; 53 and 73; 53 and 74; 53 and 75; 53 and 76; 53 and 77; 53 and 78; 53 and 79; 53 and 80; 53 and 81; 53 and 82; 53 and 83; 53 and 84;

53 and 85; 53 and 86; 53 and 87; 54 and 65; 54 and 66; 54 and 67; 54 and 68; 54 and 69; 54 and 70; 54 and 71; 54 and 72; 54 and 73; 54 and 74; 54 and 75; 54 and 76; 54 and 77; 54 and 78; 54 and 79; 54 and 80; 54 and 81; 54 and 82; 54 and 83; 54 and 84; 54 and 85; 54 and 86;

54 and 87; 55 and 65; 55 and 66; 55 and 67; 55 and 68; 55 and 69; 55 and 70; 55 and 71; 55 and 72; 55 and 73; 55 and 74; 55 and 75; 55 and 76; 55 and 77; 55 and 78; 55 and 79; 55 and 80; 55 and 81; 55 and 82; 55 and 83; 55 and 84; 55 and 85; 55 and 86; 55 and 87; 56 and 65;

56 and 66; 56 and 67; 56 and 68; 56 and 69; 56 and 70; 56 and 71; 56 and 72; 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 56 and 78; 56 and 79; 56 and 80; 56 and 81; 56 and 82; 56 and 83; 56 and 84; 56 and 85; 56 and 86; 56 and 87; 57 and 65; 57 and 66; 57 and 67;

57 and 68; 57 and 69; 57 and 70; 57 and 71; 57 and 72; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 57 and 78; 57 and 79; 57 and 80; 57 and 81; 57 and 82; 57 and 83; 57 and 84; 57 and 85; 57 and 86; 57 and 87; 58 and 65; 58 and 66; 58 and 67; 58 and 68; 58 and 69;

58 and 70; 58 and 71; 58 and 72; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 58 and 78; 58 and 79; 58 and 80; 58 and 81; 58 and 82; 58 and 83; 58 and 84; 58 and 85; 58 and 86; 58 and 87; 59 and 65; 59 and 66; 59 and 67; 59 and 68; 59 and 69; 59 and 70; 59 and 71;

59 and 72; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 59 and 78; 59 and 79; 59 and 80; 59 and 81; 59 and 82; 59 and 83; 59 and 84; 59 and 85; 59 and 86; 59 and 87; 60 and 65; 60 and 66; 60 and 67; 60 and 68; 60 and 69; 60 and 70; 60 and 71; 60 and 72; 60 and 73;

60 and 74; 60 and 75; 60 and 76; 60 and 77; 60 and 78; 60 and 79; 60 and 80; 60 and 81; 60 and 82; 60 and 83; 60 and 84; 60 and 85; 60 and 86; 60 and 87; 61 and 65; 61 and 66; 61 and 67; 61 and 68; 61 and 69; 61 and 70; 61 and 71; 61 and 72; 61 and 73; 61 and 74; 61 and 75;

61 and 76; 61 and 77; 61 and 78; 61 and 79; 61 and 80; 61 and 81; 61 and 82; 61 and 83; 61 and 84; 61 and 85; 61 and 86; 61 and 87; 62 and 65; 62 and 66; 62 and 67; 62 and 68; 62 and 69; 62 and 70; 62 and 71; 62 and 72; 62 and 73; 62 and 74; 62 and 75; 62 and 76; 62 and 77;

62 and 78; 62 and 79; 62 and 80; 62 and 81; 62 and 82; 62 and 83; 62 and 84; 62 and 85; 62 and 86; 62 and 87; 63 and 65; 63 and 66; 63 and 67; 63 and 68; 63 and 69; 63 and 70; 63 and 71; 63 and 72; 63 and 73; 63 and 74; 63 and 75; 63 and 76; 63 and 77; 63 and 78; 63 and 79;

63 and 80; 63 and 81; 63 and 82; 63 and 83; 63 and 84; 63 and 85; 63 and 86; 63 and 87; 64 and 65; 64 and 66; 64 and 67; 64 and 68; 64 and 69; 64 and 70; 64 and 71; 64 and 72; 64 and 73; 64 and 74; 64 and 75; 64 and 76; 64 and 77; 64 and 78; 64 and 79; 64 and 80; 64 and 81;

64 and 82; 64 and 83; 64 and 84; 64 and 85; 64 and 86; or 64 and 87; b. SEQ ID NOs: 88 and 65; 88 and 66; 88 and 67; 88 and 68; 88 and 69; 88 and 70; 88 and 71; 88 and 72; 88 and 73; 88 and 74; 88 and 75; 88 and 76; 88 and 77; 88 and 78; 88 and 79; 88 and 80; 88 and 81; 88 and 82; 88 and 83; 88 and 84; 88 and 85; 88 and 86; 88 and 87; 89 and 65;

89 and 66; 89 and 67; 89 and 68; 89 and 69; 89 and 70; 89 and 71; 89 and 72; 89 and 73; 89 and 74; 89 and 75; 89 and 76; 89 and 77; 89 and 78; 89 and 79; 89 and 80; 89 and 81; 89 and 82; 89 and 83; 89 and 84; 89 and 85; 89 and 86; 89 and 87; 90 and 65; 90 and 66; 90 and 67;

90 and 68; 90 and 69; 90 and 70; 90 and 71; 90 and 72; 90 and 73; 90 and 74; 90 and 75; 90 and 76; 90 and 77; 90 and 78; 90 and 79; 90 and 80; 90 and 81; 90 and 82; 90 and 83; 90 and 84; 90 and 85; 90 and 86; 90 and 87; 91 and 65; 91 and 66; 91 and 67; 91 and 68; 91 and 69;

91 and 70; 91 and 71; 91 and 72; 91 and 73; 91 and 74; 91 and 75; 91 and 76; 91 and 77; 91 and 78; 91 and 79; 91 and 80; 91 and 81; 91 and 82; 91 and 83; 91 and 84; 91 and 85; 91 and 86; 91 and 87; 92 and 65; 92 and 66; 92 and 67; 92 and 68; 92 and 69; 92 and 70; 92 and 71;

92 and 72; 92 and 73; 92 and 74; 92 and 75; 92 and 76; 92 and 77; 92 and 78; 92 and 79; 92 and 80; 92 and 81; 92 and 82; 92 and 83; 92 and 84; 92 and 85; 92 and 86; 92 and 87; 93 and 65; 93 and 66; 93 and 67; 93 and 68; 93 and 69; 93 and 70; 93 and 71; 93 and 72; 93 and 73;

93 and 74; 93 and 75; 93 and 76; 93 and 77; 93 and 78; 93 and 79; 93 and 80; 93 and 81; 93 and 82; 93 and 83; 93 and 84; 93 and 85; 93 and 86; 93 and 87; 94 and 65; 94 and 66; 94 and 67; 94 and 68; 94 and 69; 94 and 70; 94 and 71; 94 and 72; 94 and 73; 94 and 74; 94 and 75;

94 and 76; 94 and 77; 94 and 78; 94 and 79; 94 and 80; 94 and 81; 94 and 82; 94 and 83; 94 and 84; 94 and 85; 94 and 86; 94 and 87; 95 and 65; 95 and 66; 95 and 67; 95 and 68; 95 and 69; 95 and 70; 95 and 71; 95 and 72; 95 and 73; 95 and 74; 95 and 75; 95 and 76; 95 and 77;

95 and 78; 95 and 79; 95 and 80; 95 and 81; 95 and 82; 95 and 83; 95 and 84; 95 and 85; 95 and 86; 95 and 87; 96 and 65; 96 and 66; 96 and 67; 96 and 68; 96 and 69; 96 and 70; 96 and 71; 96 and 72; 96 and 73; 96 and 74; 96 and 75; 96 and 76; 96 and 77; 96 and 78; 96 and 79;

96 and 80; 96 and 81; 96 and 82; 96 and 83; 96 and 84; 96 and 85; 96 and 86; 96 and 87; 97 and 65; 97 and 66; 97 and 67; 97 and 68; 97 and 69; 97 and 70; 97 and 71; 97 and 72; 97 and 73; 97 and 74; 97 and 75; 97 and 76; 97 and 77; 97 and 78; 97 and 79; 97 and 80; 97 and 81;

97 and 82; 97 and 83; 97 and 84; 97 and 85; 97 and 86; 97 and 87; 98 and 65; 98 and 66; 98 and 67; 98 and 68; 98 and 69; 98 and 70; 98 and 71; 98 and 72; 98 and 73; 98 and 74; 98 and 75; 98 and 76; 98 and 77; 98 and 78; 98 and 79; 98 and 80; 98 and 81; 98 and 82; 98 and 83;

98 and 84; 98 and 85; 98 and 86; 98 and 87; 99 and 65; 99 and 66; 99 and 67; 99 and 68; 99 and 69; 99 and 70; 99 and 71; 99 and 72; 99 and 73; 99 and 74; 99 and 75; 99 and 76; 99 and 77; 99 and 78; 99 and 79; 99 and 80; 99 and 81; 99 and 82; 99 and 83; 99 and 84; 99 and 85;

99 and 86; 99 and 87; 100 and 65; 100 and 66; 100 and 67; 100 and 68; 100 and 69; 100 and

70; 100 and 71; 100 and 72; 100 and 73; 100 and 74; 100 and 75; 100 and 76; 100 and 77; 100 and 78; 100 and 79; 100 and 80; 100 and 81; 100 and 82; 100 and 83; 100 and 84; 100 and 85;

100 and 86; 100 and 87; 101 and 65; 101 and 66; 101 and 67; 101 and 68; 101 and 69; 101 and 70; 101 and 71; 101 and 72; 101 and 73; 101 and 74; 101 and 75; 101 and 76; 101 and 77; 101 and 78; 101 and 79; 101 and 80; 101 and 81; 101 and 82; 101 and 83; 101 and 84; 101 and 85;

101 and 86; 101 and 87; 102 and 65; 102 and 66; 102 and 67; 102 and 68; 102 and 69; 102 and 70; 102 and 71; 102 and 72; 102 and 73; 102 and 74; 102 and 75; 102 and 76; 102 and 77; 102 and 78; 102 and 79; 102 and 80; 102 and 81; 102 and 82; 102 and 83; 102 and 84; 102 and 85;

102 and 86; 102 and 87; 103 and 65; 103 and 66; 103 and 67; 103 and 68; 103 and 69; 103 and 70; 103 and 71; 103 and 72; 103 and 73; 103 and 74; 103 and 75; 103 and 76; 103 and 77; 103 and 78; 103 and 79; 103 and 80; 103 and 81; 103 and 82; 103 and 83; 103 and 84; 103 and 85;

103 and 86; 103 and 87; 104 and 65; 104 and 66; 104 and 67; 104 and 68; 104 and 69; 104 and 70; 104 and 71; 104 and 72; 104 and 73; 104 and 74; 104 and 75; 104 and 76; 104 and 77; 104 and 78; 104 and 79; 104 and 80; 104 and 81; 104 and 82; 104 and 83; 104 and 84; 104 and 85;

104 and 86; 104 and 87; 105 and 65; 105 and 66; 105 and 67; 105 and 68; 105 and 69; 105 and 70; 105 and 71; 105 and 72; 105 and 73; 105 and 74; 105 and 75; 105 and 76; 105 and 77; 105 and 78; 105 and 79; 105 and 80; 105 and 81; 105 and 82; 105 and 83; 105 and 84; 105 and 85;

105 and 86; 105 and 87; 106 and 65; 106 and 66; 106 and 67; 106 and 68; 106 and 69; 106 and 70; 106 and 71; 106 and 72; 106 and 73; 106 and 74; 106 and 75; 106 and 76; 106 and 77; 106 and 78; 106 and 79; 106 and 80; 106 and 81; 106 and 82; 106 and 83; 106 and 84; 106 and 85;

106 and 86; 106 and 87; 107 and 65; 107 and 66; 107 and 67; 107 and 68; 107 and 69; 107 and 70; 107 and 71; 107 and 72; 107 and 73; 107 and 74; 107 and 75; 107 and 76; 107 and 77; 107 and 78; 107 and 79; 107 and 80; 107 and 81; 107 and 82; 107 and 83; 107 and 84; 107 and 85;

107 and 86; 107 and 87; 108 and 65; 108 and 66; 108 and 67; 108 and 68; 108 and 69; 108 and 70; 108 and 71; 108 and 72; 108 and 73; 108 and 74; 108 and 75; 108 and 76; 108 and 77; 108 and 78; 108 and 79; 108 and 80; 108 and 81; 108 and 82; 108 and 83; 108 and 84; 108 and 85;

108 and 86; 108 and 87; 109 and 65; 109 and 66; 109 and 67; 109 and 68; 109 and 69; 109 and 70; 109 and 71; 109 and 72; 109 and 73; 109 and 74; 109 and 75; 109 and 76; 109 and 77; 109 and 78; 109 and 79; 109 and 80; 109 and 81; 109 and 82; 109 and 83; 109 and 84; 109 and 85;

109 and 86; 109 and 87; 110 and 65; 110 and 66; 110 and 67; 110 and 68; 110 and 69; 110 and 70; 110 and 71; 110 and 72; 110 and 73; 110 and 74; 110 and 75; 110 and 76; 110 and 77; 110 and 78; 110 and 79; 110 and 80; 110 and 81; 110 and 82; 110 and 83; 110 and 84; 110 and 85;

110 and 86; 110 and 87; 111 and 65; 111 and 66; 111 and 67; 111 and 68; 111 and 69; 111 and 70; 111 and 71; 111 and 72; 111 and 73; 111 and 74; 111 and 75; 111 and 76; 111 and 77; 111 and 78; 111 and 79; 111 and 80; 111 and 81; 111 and 82; 111 and 83; 111 and 84; 111 and 85;

111 and 86; 111 and 87; 112 and 65; 112 and 66; 112 and 67; 112 and 68; 112 and 69; 112 and 70; 112 and 71; 112 and 72; 112 and 73; 112 and 74; 112 and 75; 112 and 76; 112 and 77; 112 and 78; 112 and 79; 112 and 80; 112 and 81; 112 and 82; 112 and 83; 112 and 84; 112 and 85;

112 and 86; 112 and 87; 113 and 65; 113 and 66; 113 and 67; 113 and 68; 113 and 69; 113 and 70; 113 and 71; 113 and 72; 113 and 73; 113 and 74; 113 and 75; 113 and 76; 113 and 77; 113 and 78; 113 and 79; 113 and 80; 113 and 81; 113 and 82; 113 and 83; 113 and 84; 113 and 85;

113 and 86; 113 and 87; 114 and 65; 114 and 66; 114 and 67; 114 and 68; 114 and 69; 114 and 70; 114 and 71; 114 and 72; 114 and 73; 114 and 74; 114 and 75; 114 and 76; 114 and 77; 114 and 78; 114 and 79; 114 and 80; 114 and 81; 114 and 82; 114 and 83; 114 and 84; 114 and 85;

114 and 86; 114 and 87; 115 and 65; 115 and 66; 115 and 67; 115 and 68; 115 and 69; 115 and 70; 115 and 71; 115 and 72; 115 and 73; 115 and 74; 115 and 75; 115 and 76; 115 and 77; 115 and 78; 115 and 79; 115 and 80; 115 and 81; 115 and 82; 115 and 83; 115 and 84; 115 and 85;

115 and 86; 115 and 87; 116 and 65; 116 and 66; 116 and 67; 116 and 68; 116 and 69; 116 and 70; 116 and 71; 116 and 72; 116 and 73; 116 and 74; 116 and 75; 116 and 76; 116 and 77; 116 and 78; 116 and 79; 116 and 80; 116 and 81; 116 and 82; 116 and 83; 116 and 84; 116 and 85;

116 and 86; 116 and 87; 117 and 65; 117 and 66; 117 and 67; 117 and 68; 117 and 69; 117 and 70; 117 and 71; 117 and 72; 117 and 73; 117 and 74; 117 and 75; 117 and 76; 117 and 77; 117 and 78; 117 and 79; 117 and 80; 117 and 81; 117 and 82; 117 and 83; 117 and 84; 117 and 85;

117 and 86; 117 and 87; 118 and 65; 118 and 66; 118 and 67; 118 and 68; 118 and 69; 118 and 70; 118 and 71; 118 and 72; 118 and 73; 118 and 74; 118 and 75; 118 and 76; 118 and 77; 118 and 78; 118 and 79; 118 and 80; 118 and 81; 118 and 82; 118 and 83; 118 and 84; 118 and 85;

118 and 86; 118 and 87; 119 and 65; 119 and 66; 119 and 67; 119 and 68; 119 and 69; 119 and 70; 119 and 71; 119 and 72; 119 and 73; 119 and 74; 119 and 75; 119 and 76; 119 and 77; 119 and 78; 119 and 79; 119 and 80; 119 and 81; 119 and 82; 119 and 83; 119 and 84; 119 and 85;

119 and 86; 119 and 87; 120 and 65; 120 and 66; 120 and 67; 120 and 68; 120 and 69; 120 and 70; 120 and 71; 120 and 72; 120 and 73; 120 and 74; 120 and 75; 120 and 76; 120 and 77; 120 and 78; 120 and 79; 120 and 80; 120 and 81; 120 and 82; 120 and 83; 120 and 84; 120 and 85;

120 and 86; 120 and 87; 121 and 65; 121 and 66; 121 and 67; 121 and 68; 121 and 69; 121 and 70; 121 and 71; 121 and 72; 121 and 73; 121 and 74; 121 and 75; 121 and 76; 121 and 77; 121 and 78; 121 and 79; 121 and 80; 121 and 81; 121 and 82; 121 and 83; 121 and 84; 121 and 85; 121 and 86; 121 and 87; 122 and 65; 122 and 66; 122 and 67; 122 and 68; 122 and 69; 122 and 70; 122 and 71; 122 and 72; 122 and 73; 122 and 74; 122 and 75; 122 and 76; 122 and 77; 122 and 78; 122 and 79; 122 and 80; 122 and 81; 122 and 82; 122 and 83; 122 and 84; 122 and 85;

122 and 86; 122 and 87; 123 and 65; 123 and 66; 123 and 67; 123 and 68; 123 and 69; 123 and 70; 123 and 71; 123 and 72; 123 and 73; 123 and 74; 123 and 75; 123 and 76; 123 and 77; 123 and 78; 123 and 79; 123 and 80; 123 and 81; 123 and 82; 123 and 83; 123 and 84; 123 and 85;

123 and 86; 123 and 87; 124 and 65; 124 and 66; 124 and 67; 124 and 68; 124 and 69; 124 and 70; 124 and 71; 124 and 72; 124 and 73; 124 and 74; 124 and 75; 124 and 76; 124 and 77; 124 and 78; 124 and 79; 124 and 80; 124 and 81; 124 and 82; 124 and 83; 124 and 84; 124 and 85;

124 and 86; 124 and 87; 125 and 65; 125 and 66; 125 and 67; 125 and 68; 125 and 69; 125 and 70; 125 and 71; 125 and 72; 125 and 73; 125 and 74; 125 and 75; 125 and 76; 125 and 77; 125 and 78; 125 and 79; 125 and 80; 125 and 81; 125 and 82; 125 and 83; 125 and 84; 125 and 85;

125 and 86; 125 and 87; 126 and 65; 126 and 66; 126 and 67; 126 and 68; 126 and 69; 126 and 70; 126 and 71; 126 and 72; 126 and 73; 126 and 74; 126 and 75; 126 and 76; 126 and 77; 126 and 78; 126 and 79; 126 and 80; 126 and 81; 126 and 82; 126 and 83; 126 and 84; 126 and 85;

126 and 86; 126 and 87; 127 and 65; 127 and 66; 127 and 67; 127 and 68; 127 and 69; 127 and 70; 127 and 71; 127 and 72; 127 and 73; 127 and 74; 127 and 75; 127 and 76; 127 and 77; 127 and 78; 127 and 79; 127 and 80; 127 and 81; 127 and 82; 127 and 83; 127 and 84; 127 and 85;

127 and 86; 127 and 87; 128 and 65; 128 and 66; 128 and 67; 128 and 68; 128 and 69; 128 and 70; 128 and 71; 128 and 72; 128 and 73; 128 and 74; 128 and 75; 128 and 76; 128 and 77; 128 and 78; 128 and 79; 128 and 80; 128 and 81; 128 and 82; 128 and 83; 128 and 84; 128 and 85;

128 and 86; 128 and 87; 129 and 65; 129 and 66; 129 and 67; 129 and 68; 129 and 69; 129 and 70; 129 and 71; 129 and 72; 129 and 73; 129 and 74; 129 and 75; 129 and 76; 129 and 77; 129 and 78; 129 and 79; 129 and 80; 129 and 81; 129 and 82; 129 and 83; 129 and 84; 129 and 85;

129 and 86; 129 and 87; 130 and 65; 130 and 66; 130 and 67; 130 and 68; 130 and 69; 130 and 70; 130 and 71; 130 and 72; 130 and 73; 130 and 74; 130 and 75; 130 and 76; 130 and 77; 130 and 78; 130 and 79; 130 and 80; 130 and 81; 130 and 82; 130 and 83; 130 and 84; 130 and 85;

130 and 86; 130 and 87; 131 and 65; 131 and 66; 131 and 67; 131 and 68; 131 and 69; 131 and 70; 131 and 71; 131 and 72; 131 and 73; 131 and 74; 131 and 75; 131 and 76; 131 and 77; 131 and 78; 131 and 79; 131 and 80; 131 and 81; 131 and 82; 131 and 83; 131 and 84; 131 and 85;

131 and 86; 131 and 87; 132 and 65; 132 and 66; 132 and 67; 132 and 68; 132 and 69; 132 and 70; 132 and 71; 132 and 72; 132 and 73; 132 and 74; 132 and 75; 132 and 76; 132 and 77; 132 and 78; 132 and 79; 132 and 80; 132 and 81; 132 and 82; 132 and 83; 132 and 84; 132 and 85;

132 and 86; 132 and 87; 133 and 65; 133 and 66; 133 and 67; 133 and 68; 133 and 69; 133 and 70; 133 and 71; 133 and 72; 133 and 73; 133 and 74; 133 and 75; 133 and 76; 133 and 77; 133 and 78; 133 and 79; 133 and 80; 133 and 81; 133 and 82; 133 and 83; 133 and 84; 133 and 85;

133 and 86; 133 and 87; 134 and 65; 134 and 66; 134 and 67; 134 and 68; 134 and 69; 134 and 70; 134 and 71; 134 and 72; 134 and 73; 134 and 74; 134 and 75; 134 and 76; 134 and 77; 134 and 78; 134 and 79; 134 and 80; 134 and 81; 134 and 82; 134 and 83; 134 and 84; 134 and 85;

134 and 86; 134 and 87; 135 and 65; 135 and 66; 135 and 67; 135 and 68; 135 and 69; 135 and 70; 135 and 71; 135 and 72; 135 and 73; 135 and 74; 135 and 75; 135 and 76; 135 and 77; 135 and 78; 135 and 79; 135 and 80; 135 and 81; 135 and 82; 135 and 83; 135 and 84; 135 and 85;

135 and 86; 135 and 87; 136 and 65; 136 and 66; 136 and 67; 136 and 68; 136 and 69; 136 and 70; 136 and 71; 136 and 72; 136 and 73; 136 and 74; 136 and 75; 136 and 76; 136 and 77; 136 and 78; 136 and 79; 136 and 80; 136 and 81; 136 and 82; 136 and 83; 136 and 84; 136 and 85;

136 and 86; 136 and 87; 137 and 65; 137 and 66; 137 and 67; 137 and 68; 137 and 69; 137 and 70; 137 and 71; 137 and 72; 137 and 73; 137 and 74; 137 and 75; 137 and 76; 137 and 77; 137 and 78; 137 and 79; 137 and 80; 137 and 81; 137 and 82; 137 and 83; 137 and 84; 137 and 85;

137 and 86; 137 and 87; 138 and 65; 138 and 66; 138 and 67; 138 and 68; 138 and 69; 138 and 70; 138 and 71; 138 and 72; 138 and 73; 138 and 74; 138 and 75; 138 and 76; 138 and 77; 138 and 78; 138 and 79; 138 and 80; 138 and 81; 138 and 82; 138 and 83; 138 and 84; 138 and 85;

138 and 86; 138 and 87; 139 and 65; 139 and 66; 139 and 67; 139 and 68; 139 and 69; 139 and 70; 139 and 71; 139 and 72; 139 and 73; 139 and 74; 139 and 75; 139 and 76; 139 and 77; 139 and 78; 139 and 79; 139 and 80; 139 and 81; 139 and 82; 139 and 83; 139 and 84; 139 and 85;

139 and 86; 139 and 87; 140 and 65; 140 and 66; 140 and 67; 140 and 68; 140 and 69; 140 and 70; 140 and 71; 140 and 72; 140 and 73; 140 and 74; 140 and 75; 140 and 76; 140 and 77; 140 and 78; 140 and 79; 140 and 80; 140 and 81; 140 and 82; 140 and 83; 140 and 84; 140 and 85;

140 and 86; 140 and 87; 141 and 65; 141 and 66; 141 and 67; 141 and 68; 141 and 69; 141 and 70; 141 and 71; 141 and 72; 141 and 73; 141 and 74; 141 and 75; 141 and 76; 141 and 77; 141 and 78; 141 and 79; 141 and 80; 141 and 81; 141 and 82; 141 and 83; 141 and 84; 141 and 85;

141 and 86; 141 and 87; 142 and 65; 142 and 66; 142 and 67; 142 and 68; 142 and 69; 142 and 70; 142 and 71; 142 and 72; 142 and 73; 142 and 74; 142 and 75; 142 and 76; 142 and 77; 142 and 78; 142 and 79; 142 and 80; 142 and 81; 142 and 82; 142 and 83; 142 and 84; 142 and 85;

142 and 86; 142 and 87; 143 and 65; 143 and 66; 143 and 67; 143 and 68; 143 and 69; 143 and 70; 143 and 71; 143 and 72; 143 and 73; 143 and 74; 143 and 75; 143 and 76; 143 and 77; 143 and 78; 143 and 79; 143 and 80; 143 and 81; 143 and 82; 143 and 83; 143 and 84; 143 and 85;

143 and 86; or 143 and 87; c. SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and 80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70; 57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76; 58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; or d. SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82; 121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83;

121 and 83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and 84; 119 and 84; 120 and

84; 121 and 84; 90 and 84; 115 and 85; 116 and 85; 117 and 85; 118 and 85; 119 and 85; 120 and 85; 121 and 85; 90 and 85; 115 and 68; 116 and 68; 117 and 68; 118 and 68; 119 and 68;

120 and 68; 121 and 68; 90 and 68; 115 and 69; 116 and 69; 117 and 69; 118 and 69; 119 and

69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and 70; 90 and 70; 115 and 71; 116 and 71; 117 and 71; 118 and 71;

119 and 71; 120 and 71; 121 and 71; 90 and 71; 115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and 75; 116 and 75; 117 and 75;

118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76; 116 and 76; 117 and

76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and 81; 117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81.

[00128] In some embodiments, the disclosure provides for one or more nucleic acid molecules encoding a pair of guide RNAs comprising a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA target intron 52 and exon 53 of the DMD gene. In some embodiments, the guide RNAs are for use with a SluCas9 endonuclease. In some embodiments, the one or more nucleic acid molecules also encode for a SluCas9 endonuclease. In some embodiments, the one or more nucleic acid molecules are in a composition further comprising a SluCas9 endonuclease. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 2 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

36 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

37 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

38 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

35 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 35 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

36 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 36 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

39 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 37 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 38 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 39 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

19 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO:

20 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 57 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 58 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 19 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 20 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments the guide RNAs each comprise a scaffold sequence comprising the sequence of SEQ ID NO: 901.

[00129] In some embodiments, the disclosure provides for one or more nucleic acid molecules encoding a pair of guide RNAs comprising a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA target exon 53 and intron 53 of the DMD gene. In some embodiments, the guide RNAs are for use with a SluCas9 endonuclease. In some embodiments, the one or more nucleic acid molecules also encode for a SluCas9 endonuclease. In some embodiments, the one or more nucleic acid molecules are in a composition further comprising a SluCas9 endonuclease. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 82. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 83. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 84. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 85. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 68. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 69. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 70. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 71. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 73. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 74. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 75. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 76. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 80. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 116 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 117 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 118 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 119 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 120 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 121 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments, the first guide RNA comprises the sequence of SEQ ID NO: 90 and the second guide RNA comprises the sequence of SEQ ID NO: 81. In some embodiments the guide RNAs each comprise a scaffold sequence comprising the sequence of SEQ ID NO: 901. [00130] In some embodiments, a composition is provided comprising or consisting of a single nucleic acid molecule comprising: (i) a nucleic acid encoding SluCas9 and at least one, at least two, or at least three guide RNAs; or (ii) a nucleic acid encoding SluCas9 and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or (iii) a nucleic acid encoding SluCas9 and 1, 2, or 3 guide RNAs. In such embodiments, each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A-1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide sequence selected from the pairs of guide sequences shown in Table 1E or 1F, and further optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence. [00131] In some embodiments, a composition is provided comprising or consisting of two nucleic acid molecules comprising: v. a first nucleic acid encoding SluCas9; and a second nucleic acid that does not encode a SluCas9 and encodes any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; vi. a first nucleic acid encoding SluCas9 and

1. at least one, at least two, or at least three guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. 1, 2, or 3 guide RNAs; and a second nucleic acid that does not encode a SluCas9, optionally wherein the second nucleic acid comprises any one of the following:

4. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

5. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

6. from one to six guide RNAs; vii. a first nucleic acid encoding SluCas9 and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SluCas9 and encodes from one to six guide RNAs; or viii. a first nucleic acid encoding SluCas9 and at least two guide RNAs, and a second nucleic acid that does not encode a SluCas9 and encodes at least one additional copy of each of the guide RNAs encoded in the first nucleic acid; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A-1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from the pairs of guide sequences shown in Table 1E or 1F, and optionally wherein a first guide RNA of the at least one pair of guide RNAs.

C. Guide pairs targeting a splice acceptor site or a splice donor site of the DMD gene

[00132] In some embodiments, a composition is provided comprising (a) a pair of guide RNA molecules or a nucleic acid encoding a pair of RNA molecules, wherein the first guide RNA targets a region upstream of a splice acceptor site or a splice donor site in a DMD gene and wherein the second guide RNA targets a region downstream of a splice acceptor site or a splice donor site in the DMD gene, and (b) an endonuclease or a nucleic acid encoding an endonuclease. In some embodiments, the endonuclease is a Staphylococcus lugdunensis Cas9 (SluCas9). In one particular embodiment, the first guide RNA is in an intron of the DMD gene and the second guide RNA is in an exon of the DMD gene. In another particular embodiment, the first guide RNA is in an exon of the DMD gene and the second guide RNA is in an intron of the DMD gene. In another particular embodiment, the first guide RNA is in an exon of the DMD gene and the second guide RNA is in an exon of the DMD gene. In another particular embodiment, the first guide RNA is in an intron of the DMD gene and the second guide RNA is in an intron of the DMD gene.

[00133] In some embodiments, the splice acceptor site or splice donor site is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 78 of the DMD gene. In particular embodiments, the splice acceptor site or splice donor site is in intron 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57 of the DMD gene.

[00134] In some embodiments of the disclosed compositions, the first guide RNA targets a region within 1-200, 1-150, 1-100, 1-50, 30-200, 30-150, 30-100, 30-50, 75-200, 75-150, 75-100, 100-200, 100-150, or 150-200 nucleotides upstream of the splice acceptor or splice donor site. In some embodiments, the first guide RNA targets a region within 1-200, 1-150, 1-100, 1-50, 30-200, 30-150, 30-100, 30-50, 75-200, 75-150, 75-100, 100-200, 100-150, or 150-200 nucleotides downstream of the splice acceptor or splice donor site. Scaffold Sequences

[00135] Each of the guide sequences shown in Tables 1A-1F may further comprise additional nucleotides to form or encode a crRNA, e.g., using any known sequence appropriate for the Cas9 being used. In some embodiments, the crRNA comprises (5’ to 3’) at least a spacer sequence and a first complementarity domain. The first complementary domain is sufficiently complementary to a second complementarity domain, which may be part of the same molecule in the case of an sgRNA or in a tracrRNA in the case of a dual or modular gRNA, to form a duplex. See, e.g., US 2017/0007679 for detailed discussion of crRNA and gRNA domains, including first and second complementarity domains.

[00136] A single-molecule guide RNA (sgRNA) can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a singlemolecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and/or an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. In particular embodiments, the disclosure provides for an sgRNA comprising a spacer sequence and a tracrRNA sequence.

[00137] The guide RNA can be considered to comprise a scaffold sequence necessary for endonuclease binding and a spacer sequence required to bind to the genomic target sequence. In some embodiments, the guide RNA comprises any of the scaffold sequences disclosed herein and any of the spacer sequences disclosed herein. In some embodiments, the guide RNA comprises any of the scaffold sequences disclosed herein and any of the spacer sequences disclosed herein, without any nucleotides between the scaffold sequence and the spacer sequence.

An exemplary scaffold sequence suitable for use with SaCas9 to follow the guide sequence at its 3’ end is:

GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTTAAACAAGGCAAAATGCCGT GTTTATCTCGTCAACTTGTTGGCGAGA (SEQ ID NO: 500) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 500, or a sequence that differs from SEQ ID NO: 500 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00138] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3’ end is referred to as “SaScaffoldVl” and is: GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGT CAACTTGTTGGCGAGAT (SEQ ID NO: 501) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 501, or a sequence that differs from SEQ ID NO: 501 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00139] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3’ end is referred to as “SaScaffoldV2” and is:

GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTTAAACAAGGCAAAATGCCGT GTTTATCTCGTCAACTTGTTGGCGAGAT (SEQ ID NO: 502) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 502, or a sequence that differs from SEQ ID NO: 502 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00140] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3’ end is referred to as “SaScaffoldV3” and is:

GTTTAAGTACTCTGGAAACAGAATCTACTTAAACAAGGCAAAATGCCGTGTTTATCTCGT CAACTTGTTGGCGAGAT (SEQ ID NO: 503) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 503, or a sequence that differs from SEQ ID NO: 503 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00141] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3’ end is referred to as “SaScaffoldV5” and is:

GTTTCAGTACTCTGGAAACAGAATCTACTGAAACAAGGCAAAATGCCGTGTTTATCTCGT CAACTTGTTGGCGAGAT (SEQ ID NO: 504) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 504, or a sequence that differs from SEQ ID NO: 504 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00142] Two exemplary scaffold sequences suitable for use with SluCas9 to follow the guide sequence at its 3’ end are:

GTTTTAGTACTCTGGAAACAGAATCTACTGAAACAAGACAATATGTCGTGTTTATCCCAT CAATTTATTGGTGGGA (SEQ ID NO: 600), or GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTGAAACAAGACAATATGTCGT GTTTATCCCATCAATTTATTGGTGGGA (SEQ ID NO: 601) in 5’ to 3’ orientation. In some embodiments, an exemplary sequence for use with SluCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 600 or SEQ ID NO: 601, or a sequence that differs from SEQ ID NO: 600 or SEQ ID NO: 601 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00143] Another exemplary scaffold sequences suitable for use with SluCas9 to follow the guide sequence at its 3’ end is:

GTTTCAGTACTCTGGAAACAGAATCTACTGAAACAAGACAATATGTCGTGTTTATCCCAT CAATTTATTGGTGGGAT (SEQ ID NO: 901) in 5’ to 3’ orientation. In some embodiments, an exemplary sequence for use with SluCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 901, or a sequence that differs from SEQ ID NO: 901 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00144] Exemplary scaffold sequences suitable for use with SluCas9 to follow the guide sequence at its 3’ end are also shown below in the 5’ to 3’ orientation.

[00145] Table 7:

[00146] In some embodiments, the scaffold sequence suitable for use with SaCas9 to follow the guide sequence at its 3’ end is selected from any one of SEQ ID NOs: 500-504 in 5’ to 3 orientation. In some embodiments, an exemplary sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one off SEQ ID NOs: 500-504, or a sequence that differs from any one of SEQ ID NOs: 500-504 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00147] In some embodiments, the scaffold sequence suitable for use with SluCas9 to follow the guide sequence at its 3’ end is selected from any one of SEQ ID NOs: 900 or 601, or 901-917 in 5’ to 3 orientation. In some embodiments, an exemplary sequence for use with SluCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one off SEQ ID NOs: 900 or 601, or 901- 917, or a sequence that differs from any one of SEQ ID NOs: 900 or 601, or 901-917 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides. In particular embodiments, an exemplary sequence for use with SluCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 901, or a sequence that differs from any one of SEQ ID NO: 901 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00148] In some embodiments, any scaffold sequence (e.g., any of the scaffold sequences disclosed herein) is suitable for use with any sRGN disclosed herein, such as any one of sRGNl, sRGN2, sRGN3, sRGN3.1, sRGN3.2, sRGN3.3, sRGN4, or a nucleic acid molecule encoding the same. In some embodiments, a scaffold sequence suitable for use with a sRGN, such as at the 3’ end of a guide RNA sequence within an expression vector comprising the sRGN (e.g., an expression vector as shown in FIGS. 5A-5B) is selected from any one of SEQ ID NOs: 500-504, 601, or 900- 917. In some embodiments, a scaffold sequence suitable for use with a sRGN, such as at the 3’ end of a guide RNA sequence within an expression vector comprising the sRGN (e.g., an expression vector as shown in FIGS. 5A-5B) is a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 500-504, 601, or 900-917, or a sequence that differs from any one of SEQ ID NOs: 500-504, 601, or 900-917 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[00149] In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 500. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 501. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 502. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 503. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 504. In some embodiments, comprising a pair of gRNAs, one of the gRNAs comprises a sequence selected from any one of SEQ ID NOs: 500-504. In some embodiments, comprising a pair of gRNAs, both of the gRNAs comprise a sequence selected from any one of SEQ ID NOs: 500-504. In some embodiments, comprising a pair of gRNAs, the nucleotides 3’ of the guide sequence of the gRNAs are the same sequence. In some embodiments, comprising a pair of gRNAs, the nucleotides 3’ of the guide sequence of the gRNAs are different sequences.

[00150] In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 901. In some embodiments, comprising a pair of gRNAs, one of the gRNAs comprises a sequence comprising SEQ ID NO: 901. In some embodiments, comprising a pair of gRNAs, both of the gRNAs comprise a sequence comprising SEQ ID NO: 901.

[00151] In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 900. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 601. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 900. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 901. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 902. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 903. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 904. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 905. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 906. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 907. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 908. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 909. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 910. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 911. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 912. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 913. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 914. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 915. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 916. In some embodiments, the nucleic acid encoding the gRNA comprises a sequence comprising SEQ ID NO: 917. In some embodiments, comprising a pair of gRNAs, one of the gRNAs comprises a sequence selected from any one of SEQ ID NOs: 900 or 601, or 901-917. In some embodiments, comprising a pair of gRNAs, both of the gRNAs comprise a sequence selected from any one of SEQ ID NOs: 900 or 601, or 901-917. In some embodiments, comprising a pair of gRNAs, the nucleotides 3’ of the guide sequence of the gRNAs are the same sequence. In some embodiments, comprising a pair of gRNAs, the nucleotides 3’ of the guide sequence of the gRNAs are different sequences.

[00152] In some embodiments, the scaffold sequence comprises one or more alterations in the stem loop 1 as compared to the stem loop 1 of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the stem loop 2 as compared to the stem loop 2 of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the tetraloop as compared to the tetraloop of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the repeat region as compared to the repeat region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the anti-repeat region as compared to the anti-repeat region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the linker region as compared to the linker region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). See, e.g., Nishimasu et al., 2015, Cell, 162:1113-1126 for description of regions of a scaffold.

[00153] Where a tracrRNA is used, in some embodiments, it comprises (5’ to 3’) a second complementary domain and a proximal domain. In the case of a sgRNA, guide sequences together with additional nucleotides (e.g., SEQ ID Nos: 500-504 (for SaCas9), and 900 or 601, or 901-917 (for SluCas9)) form or encode a sgRNA. In some embodiments, an sgRNA comprises (5’ to 3’) at least a spacer sequence, a first complementary domain, a linking domain, a second complementary domain, and a proximal domain. A sgRNA or tracrRNA may further comprise a tail domain. The linking domain may be hairpin-forming. See, e.g., US 2017/0007679 for detailed discussion and examples of crRNA and gRNA domains, including second complementarity domains, linking domains, proximal domains, and tail domains.

[00154] In some embodiments, the disclosure provides for specific nucleic acid sequences encoding one or more guide RNA components (e.g., any of the spacer and or scaffold sequences disclosed herein). The disclosure contemplates RNA equivalents of any of the DNA sequences provided herein (i.e., in which “T”s are replaced with “U”s), or DNA equivalents of any of the RNA sequences provided herein (i.e., in which “U”s are replaced with “T”s), as well as complements (including reverse complements) of any of the sequences disclosed herein.

[00155] In some embodiments, a composition is provided comprising a guide RNA, or nucleic acid encoding a guide RNA, wherein the guide RNA further comprises a trRNA. In each composition and method embodiment described herein, the crRNA (comprising the spacer sequence) and trRNA may be associated as a single RNA (sgRNA) or may be on separate RNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.

Vectors

[00156] In any embodiment comprising a nucleic acid molecule encoding a guide RNA and/or a Cas9 (such as a SluCas9), the nucleic acid molecule may be a vector. In some embodiments, a composition is provided comprising a single nucleic acid molecule encoding at least one guide RNA and Cas9 (such as a SluCas9), wherein the nucleic acid molecule is a vector. In some embodiments, a composition is provided comprising more than one nucleic acid molecule encoding a guide RNA and Cas9 (such as a SluCas9), wherein the nucleic acid molecule is a vector. In some embodiments, a composition is provided comprising more than one nucleic acid molecule wherein one molecule encodes one or more guide RNA, and the other molecule encodes a Cas9 (such as a SluCas9) plus or minus at least one guide RNA, wherein the nucleic acid molecule is a vector.

[00157] Any type of vector, such as any of those described herein, may be used. In some embodiments, the vector is a lipid nanoparticle. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a non-integrating viral vector (i.e., that does not insert sequence from the vector into a host chromosome). In some embodiments, the viral vector is an adeno- associated virus vector (AAV), a lenti viral vector, an integrase-deficient lenti viral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the vector comprises a muscle-specific promoter. Exemplary muscle-specific promoters include a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, or an SPc5-12 promoter. See US 2004/0175727 Al; Wang et al., Expert Opin Drug Deliv. (2014) 11, 345- 364; Wang et al., Gene Therapy (2008) 15, 1489-1499. In some embodiments, the muscle-specific promoter is a CK8 promoter. In some embodiments, the muscle-specific promoter is a CK8e promoter. In any of the foregoing embodiments, the vector may be an adeno-associated virus vector (AAV).

[00158] In some embodiments, the vector is a lipid nanoparticle comprising an endonuclease (e.g., any of the endonucleases disclosed herein) and one or more of any of the guide RNAs disclosed herein. In some embodiments, the vector is a lipid nanoparticle comprising a nucleic acid encoding an endonuclease (e.g., any of the endonucleases disclosed herein) and one or more of any of the guide RNAs disclosed herein. In some embodiments, the vector is a lipid nanoparticle comprising a nucleic acid encoding an endonuclease (e.g., any of the endonucleases disclosed herein) and a nucleic acid encoding one or more of any of the guide RNAs disclosed herein.

[00159] Where a vector is used, it may be a viral vector, such as a non-integrating viral vector. In some embodiments, the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO (see, e.g., SEQ ID NO: 81 of US 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74 (see, e.g., SEQ ID NO: 1 of US 2015/0111955, which is incorporated by reference herein in its entirety), or AAV9 vector, wherein the number following AAV indicates the AAV serotype. In some embodiments, the AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the AAV vector is a double-stranded AAV (dsAAV). Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. See, e.g., McCarty et al., Gene Ther. 2001;8:1248-54, Naso et al., BioDrugs 2017; 31:317-334, and references cited therein for detailed discussion of various AAV vectors. In some embodiments, the AAV vector size is measured in length of nucleotides from ITR to ITR, inclusive of both ITRs. In some embodiments, the AAV vector is less than 5 kb in size from ITR to ITR, inclusive of both ITRs. In particular embodiments, the AAV vector is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.85 kb in size from ITR to ITR, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.8 kb in size from ITR to ITR, inclusive of both

ITRs. In further embodiments, the AAV vector is less than 4.75 kb in size from ITR to ITR, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.7 kb in size from ITR to ITR, inclusive of both ITRs. In some embodiments, the vector is between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to

ITR in size, inclusive of both ITRs. In some embodiments, the vector is between 4.4-4.85 kb in size from ITR to ITR, inclusive of both ITRs. In some embodiments, the vector is an AAV9 vector.

[00160] In some embodiments, the vector (e.g., viral vector, such as an adeno-associated viral vector) comprises a tissue-specific (e.g., muscle-specific) promoter, e.g., which is operatively linked to a sequence encoding the guide RNA and/or the Cas protein. In some embodiments, the musclespecific promoter is a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, or an SPc5-12 promoter. In some embodiments, the muscle-specific promoter is a CK8 promoter. In some embodiments, the muscle-specific promoter is a CK8e promoter. Muscle-specific promoters are described in detail, e.g., in US2004/0175727 Al; Wang et al., Expert Opin Drug Deliv. (2014) 11,

345-364; Wang et al., Gene Therapy (2008) 15, 1489-1499. In some embodiments, the tissue-specific promoter is a neuron-specific promoter, such as an enolase promoter. See, e.g., Naso et al., BioDrugs

2017; 31:317-334; Dashkoff et al., Mol Ther Methods Clin Dev. 2016;3:16081, and references cited therein for detailed discussion of tissue-specific promoters including neuron-specific promoters.

[00161] In some embodiments, in addition to guide RNA and Cas9 (such as a SluCas9) sequences, the vectors further comprise nucleic acids that do not encode guide RNAs. Nucleic acids that do not encode guide RNA and Cas9 (such as a SluCas9) include, but are not limited to, promoters, enhancers, and regulatory sequences. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the muscle specific promoter is the CK8 promoter. The CK8 promoter has the following sequence

(SEQ ID NO. 700):

1 CTAGACTAGC ATGCTGCCCA TGTAAGGAGG CAAGGCCTGG GGACACCCGA GATGCCTGGT 61 TATAATTAAC CCAGACATGT GGCTGCCCCC CCCCCCCCAA CACCTGCTGC CTCTAAAAAT 121 AACCCTGCAT GCCATGTTCC CGGCGAAGGG CCAGCTGTCC CCCGCCAGCT AGACTCAGCA 181 CTTAGTTTAG GAACCAGTGA GCAAGTCAGC CCTTGGGGCA GCCCATACAA GGCCATGGGG 241 CTGGGCAAGC TGCACGCCTG GGTCCGGGGT GGGCACGGTG CCCGGGCAAC GAGCTGAAAG 301 CTCATCTGCT CTCAGGGGCC CCTCCCTGGG GACAGCCCCT CCTGGCTAGT CACACCCTGT 361 AGGCTCCTCT ATATAACCCA GGGGCACAGG GGCTGCCCTC ATTCTACCAC CACCTCCACA 421 GCACAGACAG ACACTCAGGA GCCAGCCAGC

[00162] In some embodiments, the muscle-cell cell specific promoter is a variant of the CK8 promoter, called CK8e. In some embodiments, the size of the CK8e promoter is 436 bp. The CK8e promoter has the following sequence (SEQ ID NO. 701): 1 TGCCCATGTA AGGAGGCAAG GCCTGGGGAC ACCCGAGATG CCTGGTTATA ATTAACCCAG

61 ACATGTGGCT GCCCCCCCCC CCCCAACACC TGCTGCCTCT AAAAATAACC CTGCATGCCA

121 TGTTCCCGGC GAAGGGCCAG CTGTCCCCCG CCAGCTAGAC TCAGCACTTA GTTTAGGAAC

181 CAGTGAGCAA GTCAGCCCTT GGGGCAGCCC ATACAAGGCC ATGGGGCTGG GCAAGCTGCA

241 CGCCTGGGTC CGGGGTGGGC ACGGTGCCCG GGCAACGAGC TGAAAGCTCA TCTGCTCTCA

301 GGGGCCCCTC CCTGGGGACA GCCCCTCCTG GCTAGTCACA CCCTGTAGGC TCCTCTATAT

361 AACCCAGGGG CACAGGGGCT GCCCTCATTC TACCACCACC TCCACAGCAC AGACAGACAC

421 TCAGGAGCCA GCCAGC

[00163] In some embodiments, the Ck8e promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 701.

[00164] In some embodiments, the vector comprises one or more of a U6, Hl, or 7SK promoter. In some embodiments, the U6 promoter is the human U6 promoter (e.g., the U6L promoter or U6S promoter). In some embodiments, the promoter is the murine U6 promoter. In some embodiments, the 7SK promoter is a human 7SK promoter. In some embodiments, the 7SK promoter is the 7SK1 promoter. In some embodiments, the 7SK promoter is the 7SK2 promoter. In some embodiments, the Hl promoter is a human Hl promoter (e.g., the H1L promoter or the HIS promoter). In some embodiments, the vector comprises multiple guide sequences, wherein each guide sequence is under the control of a separate promoter. In some embodiments, each of the multiple guide sequences comprises a different sequence. In some embodiments, each of the multiple guide sequences comprise the same sequence (e.g., each of the multiple guide sequences comprise the same spacer sequence). In some embodiments, each of the multiple guide sequences comprises the same spacer sequence and the same scaffold sequence. In some embodiments, each of the multiple guide sequences comprises different spacer sequences and different scaffold sequences. In some embodiments, each of the multiple guide sequences comprises the same spacer sequence, but comprises a different scaffold sequence. In some embodiments, each of the multiple guide sequences comprises different spacer sequences and different scaffold sequences. In some embodiments, each of the separate promoters comprises the same nucleotide sequence (e.g., the U6 promoter sequence). In some embodiments, each of the separate promoters comprises a different nucleotide sequence e.g., the U6, Hl, and/or 7SK promoter sequence).

[00165] In some embodiments, the U6 promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 702: cgagtccaac acccgtggga atcccatggg caccatggcc cctcgctcca aaaatgcttt 60 cgcgtcgcgc agacactgct cggtagtttc ggggatcagc gtttgagtaa gagcccgcgt 120 ctgaaccctc cgcgccgccc cggccccagt ggaaagacgc gcaggcaaaa cgcaccacgt 180 gacggagcgt gaccgcgcgc cgagcgcgcg ccaaggtcgg gcaggaagag ggcctatttc 240 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta 300 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc 360 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa 420 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aa 472 [00166] In some embodiments, the Hl promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 703: gctcggcgcg cccatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa 60 atgtctttgg atttgggaat cttataagtt ctgtatgaga ccacggta 108

[00167] In some embodiments, the 7SK promoter comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ

ID NO: 704: tgacggcgcg ccctgcagta tttagcatgc cccacccatc tgcaaggcat tctggatagt 60 gtcaaaacag ccggaaatca agtccgttta tctcaaactt tagcattttg ggaataaatg 120 atatttgcta tgctggttaa attagatttt agttaaattt cctgctgaag ctctagtacg 180 ataagtaact tgacctaagt gtaaagttga gatttccttc aggtttatat agcttgtgcg 240 ccgcctgggt a

251

[00168] In some embodiments, the U6 promoter is a hU6c promoter and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 705: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAG AAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCAT ATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC GAAACACC.

[00169] In some embodiments, the U6 promoter is a variant of the hU6c promoter. In some embodiments, the variant of the hU6c promoter comprises alternative nucleotides as compared to the sequence of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter comprises fewer nucleotides as compared to the 249 nucleotides of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter has fewer nucleotides in the nucleosome binding sequence of the hU6c promoter of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, or 30 nucleotides) the nucleotides corresponding to nucleotides 96-125 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides) the nucleotides corresponding to nucleotides 81-140 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, or 85 nucleotides) the nucleotides corresponding to nucleotides 66- 150 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides) the nucleotides corresponding to nucleotides 51-170 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks the nucleotides corresponding to nucleotides 96-125 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter comprises 129-219 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 219 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 189 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 159 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 129 nucleotides.

[00170] In some embodiments, the U6 promoter is hU6d30 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9001: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATTGGAATTAATTTGACTGTAAACACAAAGATATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC.

[00171] In some embodiments, the U6 promoter is hU6d60 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9002: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATTGGAATTAATTTGACGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATA TGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACG AAACACC.

[00172] In some embodiments, the U6 promoter is hU6d90 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9003: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC.

[00173] In some embodiments, the U6 promoter is hU6dl20 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9004:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCGGACTATCAT ATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC GAAACACC.

[00174] In some embodiments, the 7SK promoter is a 7SK2 promoter and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 706: CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC GGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATG CTGGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGCAACTTG ACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGTAC

CTC.

[00175] In some embodiments, the 7SK promoter is a variant of the 7SK2 promoter. In some embodiments, the variant of the 7SK2 promoter comprises alternative nucleotides as compared to the sequence of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter e.g., comprises fewer nucleotides as compared to the 243 nucleotides of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter has fewer nucleotides in the nucleosome binding sequence of the 7SK2 promoter of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, or 30 nucleotides) the nucleotides corresponding to nucleotides 95-124 of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides) the nucleotides corresponding to nucleotides 81-140 of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85 or 90 nucleotides) the nucleotides corresponding to nucleotides 67-156 of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides) the nucleotides corresponding to nucleotides 52-171 of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter comprises 123-213 nucleotides. In some embodiments, the variant of the 7SK2 promoter comprises 213 nucleotides. In some embodiments, the variant of the 7SK2 promoter comprises 183 nucleotides. In some embodiments, the variant of the 7SK2 promoter comprises 153 nucleotides. In some embodiments, the variant of the 7SK2 promoter comprises 123 nucleotides.

[00176] In some embodiments, the 7SK promoter is 7SKd30 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9006: CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC GGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTAAATTAGATTTTAGTTAAATTTCCT GCTGAAGCTCTAGTACGATAAGCAACTTGACCTAAGTGTAAAGTTGAGACTTCCTTCAGG TTTATATAGCTTGTGCGCCGCTTGGGTACCTC.

[00177] In some embodiments, the 7SK promoter is 7SKd60 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9007: CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC GGAAATCAAGTCCGTTTATCTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGCAACTTG ACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGTAC CTC. [00178] In some embodiments, the 7SK promoter is 7SKd90 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9008:

CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC GGAAATAGCTCTAGTACGATAAGCAACTTGACCTAAGTGTAAAGTTGAGACTTCCTTCAG GTTTATATAGCTTGTGCGCCGCTTGGGTACCTC.

[00179] In some embodiments, the 7SK promoter is 7SKdl20 and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9009:

[00180] CTGC AGTATTTAGC ATGCCCCACCC ATCTGCA AGGCATTCTGGATAGTGTC AG

CAACTTGACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTT GGGTACCTC.In some embodiments, the Hl promoter is a Him or mHl promoter and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 707:

AATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGAT TTGGGAATCTTATAAGTTCTGTATGAGACCACTCTTTCCC.

[00181] In some embodiments, the promoter is an Ml 1 promoter and comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 708:

ATATTTAGCATGTCGCTATGTGTTCTGGGAAACTTGACCTAAGTGTAAAGTTGAGATTTC CTTCAGGTTTATATAGTTCTGTATGAGACCACTCTTTCCC.

[00182] In some embodiments, the vector comprises multiple inverted terminal repeats (ITRs). These ITRs may be of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some embodiments, the ITRs are of an AAV2 serotype. In some embodiments, the 5’ ITR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 709:

GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG CCAACTCCATCACTAGGGGTTCCT.

[00183] In some embodiments, the 5’ ITR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 939:

CGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC GACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTG GCCAACTCCATCACTAGGGGTTCCT. [00184] In some embodiments, the 3’ITR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 710:

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGG CCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGA.

[00185] In some embodiments, the 3’ ITR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 940:

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGG CCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGAA.

[00186] In some embodiments, a vector comprising a single nucleic acid molecule encoding 1) two or more guide RNA comprising any one or more of the spacer sequences of Table s 1A-1F ; and 2) a SluCas9 is provided. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is administered to a subject to treat DMD. In some embodiments, only one vector is needed due to the use of a particular guide sequence that is useful in the context of SaCas9 or SluCas9.

[00187] In some embodiments, the vector comprises a nucleic acid encoding a Cas9 protein (e.g., an SaCas9 or SluCas9 protein) and further comprises a nucleic acid encoding one or more single guide RNA(s). In some embodiments, the nucleic acid encoding the Cas9 (such as a SluCas9) protein is under the control of a CK8e promoter. In some embodiments, the nucleic acid encoding the guide RNA sequence is under the control of a hU6c promoter. In some embodiments, the vector is AAV9.

[00188] In some embodiments, the vector comprises multiple nucleic acids encoding more than one guide RNA. In some embodiments, the vector comprises two nucleic acids encoding two different guide RNA sequences.

[00189] In some embodiments, the vector comprises a nucleic acid encoding a Cas9 protein (such as a SluCas9), a nucleic acid encoding a first guide RNA, and a nucleic acid encoding a second guide RNA. In some embodiments, the vector does not comprise a nucleic acid encoding more than two guide RNAs. In some embodiments, the vector comprises a single nucleic acid molecule, wherein the single nucleic acid molecule comprises a nucleic acid encoding a Cas9 (such as a SluCas9) protein, a nucleic acid encoding a first guide RNA, and a nucleic acid that is the reverse complement to the coding sequence for the second guide RNA. In some embodiments, the vector comprises a single nucleic acid molecule, wherein the single nucleic acid molecule comprises a nucleic acid encoding a Cas9 (such as a SluCas9) protein, a nucleic acid that is the reverse complement to the coding sequence for the first guide RNA, and a nucleic acid that is the reverse complement to the coding sequence for the second guide RNA. In some embodiments, the nucleic acid encoding a Cas9 protein (such as a SluCas9) is under the control of the CK8e promoter. In some embodiments, the first guide is under the control of the hU6c promoter and the second guide is under the control of the hU6c promoter. In some embodiments, the first guide is under the control of the 7SK2 promoter, and the second guide is under the control of the Him promoter. In some embodiments, the first guide is under the control of the Him promoter, and the second guide is under the control of the 7SK2 promoter. In some embodiments, the first guide is under the control of the hU6c promoter, and the second guide is under the control of the Him promoter. In some embodiments, the first guide is under the control of the Him promoter, and the second guide is under the control of the hU6c promoter. In some embodiments, the nucleic acid encoding the Cas9 protein is: a) between the nucleic acids encoding the guide RNAs, b) between the nucleic acids that are the reverse complement to the coding sequences for the guide RNAs, c) between the nucleic acid encoding the first guide RNA and the nucleic acid that is the reverse complement to the coding sequence for the second guide RNA, d) between the nucleic acid encoding the second guide RNA and the nucleic acid that is the reverse complement to the coding sequence for the first guide RNA, e) 5’ to the nucleic acids encoding the guide RNAs, f) 5’ to the nucleic acids that are the reverse complements to the coding sequences for the guide RNAs, g) 5’ to a nucleic acid encoding one of the guide RNAs and 5’ to a nucleic acid that is the reverse complement to the coding sequence for the other guide RNA, h) 3’ to the nucleic acids encoding the guide RNAs, i) 3’ to the nucleic acids that are the reverse complements to the coding sequences for the guide RNAs, or j) 3’ to a nucleic acid encoding one of the guide RNAs and 3’ to a nucleic acid that is the reverse complement to the coding sequence for the other guide RNA. In some embodiments, any of the vectors disclosed herein is AAV9. In preferred embodiments, the AAV9 vector is less than 5 kb from ITR to ITR in size, inclusive of both ITRs. In particular embodiments, the AAV9 vector is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.85 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.8 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.7 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the vector is between 3.9- 5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9- 4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the vector is between 4.4-4.85 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the vector is an AAV9 vector.

[00190] In some embodiments, any of the vectors disclosed herein comprises a nucleic acid encoding at least a first guide RNA and a second guide RNA. In some embodiments, the nucleic acid comprises a spacer-encoding sequence for the first guide RNA, a scaffold-encoding sequence for the first guide RNA, a spacer-encoding sequence for the second guide RNA, and a scaffold-encoding sequence of the second guide RNA. In some embodiments, the scaffold-encoding sequence for the first guide RNA is identical to the scaffold-encoding sequence for the second guide RNA. In some embodiments, the scaffold-encoding sequence for the first guide RNA is different from the scaffoldencoding sequence for the nucleic acid encoding the second guide RNA. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises a sequence selected from the group consisting of SEQ ID Nos: 500-504 (for SaCas9) and 601 or 900-917 (for SluCas9), and the scaffoldencoding sequence for the second guide RNA comprises a different sequence selected from the group consisting of SEQ ID Nos: 500-504 (for SaCas9) and 601 or 900-917 (for SluCas9). In particular embodiments, the scaffold-encoding sequence for the first guide RNA comprises a sequence comprising the sequence of SEQ ID NO: 901. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 901. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 500, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 501. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 500, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 502. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 500, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 503. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 500, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 504. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 501, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 502. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 501, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 503. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 501, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 504. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 502, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 503. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 502, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 504. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 503, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 504. In particular embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 504, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 504. In some embodiments, the scaffoldencoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 900. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 901. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 902. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 903. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 904. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 905. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 906. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 907. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 908. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 909. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 910. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 911. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 912. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 913. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 914. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 915. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 916. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 600, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 917. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 902. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 903. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 904. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 905. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 906. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 907. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 908. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 909. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 910. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 911. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 912. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 913. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 914. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 915. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffoldencoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 916. In some embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 917. In particular embodiments, the scaffold-encoding sequence for the first guide RNA comprises the sequence of SEQ ID NO: 901, and the scaffold-encoding sequence for the second guide RNA comprises the sequence of SEQ ID NO: 901. In some embodiments, the spacer encoding sequence for the first guide RNA is the same as the spacer-encoding sequence in the second guide RNA, and the scaffold-encoding sequence for the first guide RNA is different from the scaffoldencoding sequence in the nucleic acid encoding the second guide RNA.

[00191] In some embodiments, the AAV vector is in a particular configuration. Some examples of these AAV vector configurations are provided herein, and the order of elements in these exemplary vectors are referenced in a 5’ to 3’ manner with respect to the plus strand. For these configurations, it should be understood that the recited elements may not be directly contiguous, and that one or more nucleotides or one or more additional elements may be present between the recited elements. However, in some embodiments, it is possible that no nucleotides or no additional elements are present between the recited elements. Also, unless otherwise stated, “a promoter for expression of element X” means that the promoter is oriented in a manner to facilitate expression of the recited element X. In addition, unless otherwise stated, references to an “sgRNA scaffold sequence” or “a guide RNA scaffold sequence” are synonymous with “a nucleotide sequence/nucleic acid encoding an sgRNA scaffold sequence” or “a nucleotide sequence/nucleic acid encoding a guide RNA scaffold sequence.” In some embodiments, the disclosure provides for a nucleic acid encoding an SaCas9 (e.g., an SaCas9-KKH) or SluCas9. In some embodiments, the nucleic acid encodes for one or more nuclear localization signals (e.g., the SV40 NLS and/or the c-Myc NLS) on the C-terminus of the encoded SaCas9 or SluCas9. In some embodiments, the nucleic acid encodes for one or more NLSs (e.g., the SV40 NLS and/or the c-Myc NLS) on the C-terminus of the encoded SaCas9 or SluCas9, and the nucleic acid does not encode for an NLS on the N-terminus of the encoded SaCas9 or SluCas9. In some embodiments, the nucleic acid encodes for one or more nuclear localization signals (e.g., the SV40 NLS and/or the c-Myc NLS) on the N-terminus of the encoded SaCas9 or SluCas9. In some embodiments, the nucleic acid encodes for one or more NLSs (e.g., the SV40 NLS and/or the c- Myc NLS) on the N-terminus of the encoded SaCas9 or SluCas9, and the nucleic acid does not encode for an NLS on the C-terminus of the encoded SaCas9 or SluCas9. In some embodiments, the nucleic acid encodes for one or more nuclear localization signals (e.g., the SV40 NLS and/or the c- Myc NLS) on the C-terminus of the encoded SaCas9 or SluCas9 and also encodes for one or more NLSs on the N-terminus of the encoded SaCas9 or SluCas9 (e.g., the SV40 NLS and/or the c-Myc NLS). In some embodiments, the nucleic acid encodes one NLS. In some embodiments, the nucleic acid encodes two NLSs. In some embodiments, the nucleic acid encodes three NLSs. The one, two, or three NLS may all be C-terminal, N-terminal, or any combination of C- and N-terminal. The NLS may be fused/attached directly to the C- or N-terminus or to another NLS, or may be fused/attached indirectly attached through a linker. In some embodiments, an additional domain may be: a) fused to the N- or C-terminus of the Cas protein (e.g., a Cas9 protein), b) fused to the N-terminus of an NLS fused to the N-terminus of a Cas protein, or c) fused to the C-terminus of an NLS fused to the C- terminus of a Cas protein, with or without a linker. In some embodiments, an NLS is fused to the N- and/or C-terminus of the Cas protein by means of a linker. In some embodiments, an NLS is fused to the N-terminus of an N-terminally-fused NLS on a Cas protein by means of a linker, and/or an NLS is fused to the C-terminus of a C-terminally fused NLS on a Cas protein by means of a linker. In some embodiments, the linker is GSVD (SEQ ID NO: 550) or GSGS (SEQ ID NO: 551). In some embodiments, the Cas protein comprises a c-Myc NLS fused to the N-terminus of the Cas protein (or to an N-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises an SV40 NLS fused to the C-terminus of the Cas protein (or to a C-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises a nucleoplasmin NLS fused to the C-terminus of the Cas protein (or to a C-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N-terminus of the Cas protein, optionally by means of a linker, b) an SV40 NLS fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) a nucleoplasmin NLS fused to the C-terminus of the SV40 NLS, optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N-terminus of the Cas protein, optionally by means of a linker, b) a nucleoplasmin NLS fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) an SV40 NLS fused to the C-terminus of the nucleoplasmin NLS, optionally by means of a linker. In some embodiments, a c-myc NLS is fused to the N-terminus of the Cas and an SV40 NLS and/or nucleoplasmin NLS is fused to the C-terminus of the Cas. In some embodiments, a c-myc NLS is fused to the N-terminus of the Cas (e.g., by means of a linker such as GSVD), an SV40 NLS is fused to the C-terminus of the Cas (e.g., by means of a linker such as GSGS), and a nucleoplasmin NLS is fused to the C-terminus of the SV-40 NLS (e.g., by means of a linker such as GSGS).

[00192] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of a promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9, a polyadenylation sequence, a promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA is associated with weaker expression than the second sgRNA when compared individually in a sgRNA expression assay, e.g., in an assay in which each guide is separately assessed (i.e., not in the same construct) using the same promoter, same concentration of genetic material/vector/RNP in substantially the same conditions (e.g., time, pH, temperature, buffer conditions). In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA is any of the hU6c promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9001. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9002. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9003. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9004. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA is any of the 7SK2 promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9006. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9007. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9008. In some embodiments the promoter for expression of the nucleic acid encoding the first sgRNA comprises SEQ ID NO: 9009. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the hU6c promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9001. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9002. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9003. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9004. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the 7SK2 promoters disclosed herein. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 705. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9006. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9007. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9008. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA comprises SEQ ID NO: 9009. In some embodiments the promoter for expression of the nucleic acid encoding the second sgRNA is any of the Him promoters disclosed herein. In some embodiments, the promoter for SaCas9 is the CK8e promoter. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an SV40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the one or more NLSs is a nucleoplasmin NLS. In some embodiments, the NLS is fused to the SaCas9 with a linker.

[00193] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9, a polyadenylation sequence, an hU6c promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA are selected from Tables 1A-1F. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an SV40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS is fused to the SaCas9 with a linker.

[00194] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding SaCas9 (e.g., CK8e), a nucleic acid encoding SaCas9, a polyadenylation sequence, an 7SK promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA are selected from Tables 1A-1F. In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding SaCas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an SV40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS is fused to the SaCas9 with a linker.

[00195] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an hU6c promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding Cas9 (e.g., CK8e), a nucleic acid encoding Cas9, a polyadenylation sequence, an Him promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA are selected from Tables 1A and 1F. In some embodiments, the nucleic acid sequence encoding Cas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding Cas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding Cas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an SV40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS is fused to the Cas9 with a linker.

[00196] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of an 7SK promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding Cas9 (e.g., CK8e), a nucleic acid encoding Cas9, a polyadenylation sequence, an Him promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. In some embodiments, the first sgRNA and the second sgRNA are selected from Tables 1A-1F. In some embodiments, the nucleic acid sequence encoding Cas9 is fused to a nucleic acid sequence encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding Cas9 is fused to two nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the nucleic acid sequence encoding Cas9 is fused to three nucleic acid sequences each encoding a nuclear localization sequence (NLS). In some embodiments, the one or more NLSs is an SV40 NLS. In some embodiments, the one or more NLSs is a c-Myc NLS. In some embodiments, the NLS is fused to the Cas9 with a linker.

[00197] In some embodiments, the disclosure provides for a composition comprising at least two nucleic acids. In some embodiments, the composition comprises at least two nucleic acid molecules, wherein the first nucleic acid molecule comprises a sequence encoding an endonuclease (such as a SluCas9) wherein the second nucleic acid molecule encodes a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA are not the same sequence, and wherein the second nucleic acid molecule does not encode an endonuclease. In some embodiments, the first nucleic acid molecule also encodes a copy of the first guide RNA and a copy of the second guide RNA. In some embodiments, the first nucleic acid molecule does not encode any guide RNAs. In some embodiments, the second nucleic acid molecule encodes two copies of the first guide RNA and two copies of the second guide RNA. In some embodiments, the second nucleic molecule encodes two copies of the first guide RNA, and one copy of the second guide RNA. In some embodiments, the second nucleic acid molecule encodes one copy of the first guide RNA, and two copies of the second guide RNA. In some embodiments, the second nucleic acid molecule comprises two copies of the first guide RNA, and three copies of the second guide RNA. In some embodiments, the second nucleic acid molecule comprises three copies of the first guide RNA, and two copies of the second guide RNA. In some embodiments, the second nucleic acid does not encode a Cas protein. In some embodiments, the second nucleic acid molecule encodes three copies of the first guide RNA and three copies of the second guide RNA. In some embodiments, the first nucleic acid molecule comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first guide RNA scaffold sequence, the reverse complement of a nucleotide sequence encoding the first guide RNA sequence, the reverse complement of a promoter for expression of the nucleotide sequence encoding the first guide RNA sequence, a promoter for expression of a nucleotide sequence encoding the endonuclease, a nucleotide sequence encoding an endonuclease, a polyadenylation sequence, a promoter for expression of the second guide RNA in the same direction as the promoter for the endonuclease, the second guide RNA sequence, and a second guide RNA scaffold sequence. In some embodiments, the promoter for expression of the nucleotide sequence encoding the first guide RNA sequence in the first nucleic acid molecule is a U6 promoter and the promoter for expression of the nucleotide sequence encoding the second guide RNA in the first nucleic acid molecule is a U6 promoter.

[00198] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: a promoter for expression of a nucleic acid encoding a first sgRNA, a nucleic acid encoding the first sgRNA guide sequence, the first sgRNA scaffold sequence, a promoter for expression of Cas9 (e.g., CK8e), a nucleic acid encoding Cas9 (such as SluCas9), a polyadenylation sequence, a promoter for expression of a second sgRNA, the second sgRNA guide sequence, and a second sgRNA scaffold sequence.

[00199] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first sgRNA scaffold sequence, the reverse complement of a nucleic acid encoding a first sgRNA guide sequence, the reverse complement of a promoter for expression of the nucleic acid encoding the first sgRNA, a promoter for expression of a nucleic acid encoding Cas9 (e.g., CK8e), a nucleic acid encoding Cas9 (such as SluCas9), a polyadenylation sequence, a promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence. [00200] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: a promoter for expression of a nucleic acid encoding a first sgRNA, a nucleic acid encoding the first sgRNA guide sequence, a first sgRNA scaffold sequence, a promoter for expression of a second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence, a promoter for Cas9 (e.g., CK8e), a nucleic acid encoding Cas9 (such as SluCas9), and a polyadenylation sequence.

[00201] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: a promoter for expression of a nucleic acid encoding Cas9 (e.g., CK8e), a nucleic acid encoding Cas9 (such as SluCas9), a polyadenylation sequence, a promoter for expression of the nucleic acid encoding a first guide RNA, a nucleic acid encoding the first sgRNA guide sequence, a first sgRNA scaffold sequence, a promoter for expression of the second sgRNA, a second sgRNA guide sequence, and a second sgRNA scaffold sequence.

[00202] In some embodiments, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: a first sgRNA scaffold sequence (e.g., V5), a nucleic acid encoding a first sgRNA guide sequence (e.g., Slu3 or Slu7), a promoter (e.g., U6) for expression of the nucleic acid encoding the first sgRNA guide sequence, a promoter (e.g., CK8e) for expression (in the opposite direction of expression of the nucleic acid encoding the first sgRNA guide sequence) of a nucleic acid encoding a SluCas9, a nucleic acid encoding a first NLS, a nucleic acid encoding a SluCas9, a nucleic acid encoding a second NLS, a nucleic acid encoding a third NLS, a promoter (e.g., U6) for expression (in the same direction as expression of the nucleic acid encoding the SluCas9) of a second sgRNA guide sequence (e.g., Siu 7 or Siu 3), a nucleic acid encoding the second sgRNA guide sequence, and a second sgRNA scaffold sequence (e.g., V5).

[00203] In a particular embodiment, the AAV vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first scaffold sequence (such as the scaffold sequence of SEQ ID NO: 901), the reverse complement of a nucleic acid encoding a guide RNA sequence selected from SEQ ID NOs: 1-143, the reverse complement of a U6 promoter for expression of the nucleic acid encoding the guide RNA sequence, a CK8e promoter for expression (in the opposite direction of expression of the nucleic acid encoding the sgRNA guide sequence) of a nucleic acid encoding a SluCas9, a nucleic acid encoding a c-Myc NLS, a nucleic acid encoding a SluCas9, a nucleic acid encoding a SV40 NLS, a nucleic acid encoding a NP NLS, a U6 promoter for expression (in the same direction as expression of the nucleic acid encoding the SluCas9) of a guide RNA sequence of any one of SEQ ID NOs: 1-143, a nucleic acid encoding the guide RNA sequence, and a second V5 scaffold sequence (such as the scaffold sequence of SEQ ID NO: 901).

[00204] In some embodiments, the first nucleic acid molecule is in a first vector (e.g., AAV9), and the second nucleic acid is in a separate second vector. In some embodiments, the first vector is AAV9. In some embodiments, the second vector is AAV9. In preferred embodiments, the AAV9 vector is less than 5 kb from ITR to ITR in size, inclusive of both ITRs. In particular embodiments, the AAV9 vector is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.85 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.8 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 vector is less than 4.7 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the second vector comprises from 5’ to 3’ with respect to the plus strand: a promoter for expression of a first copy of a first guide RNA (e.g., a U6 promoter), a first copy of a nucleotide sequence encoding a first guide RNA, a first copy of a nucleotide sequence encoding a first guide RNA scaffold, a promoter for expression of a second copy of the first guide RNA (e.g., a Hl promoter), a second copy of the nucleotide sequence encoding the first guide RNA, a second copy of the nucleotide sequence encoding the first guide RNA scaffold, a promoter for expression of a second guide RNA (e.g., a 7SK promoter), a nucleotide sequence encoding a second guide RNA, and a nucleotide sequence encoding a second guide RNA scaffold. In some embodiments, the second vector comprises from 5’ to 3’ with respect to the plus strand: a promoter for expression of a first guide RNA (e.g., a U6 promoter), a nucleotide sequence encoding a first guide RNA, a nucleotide sequence encoding a first guide RNA scaffold, a promoter for expression of a second guide RNA (e.g., a 7SK promoter), a nucleotide sequence encoding a second guide RNA, and a nucleotide sequence encoding a second guide RNA scaffold. In some embodiments, the second vector comprises a staffer sequence (e.g., a 3’UTR desmin sequence) between the nucleotide sequence encoding the first guide scaffold sequence and the promoter for expression of the second guide sequence. In some embodiments, the second vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a scaffold for a first guide RNA, the reverse complement of a nucleotide sequence encoding a first guide RNA, the reverse complement of a promoter for expression of the first guide RNA (e.g., a U6c promoter), a promoter for expression of a second guide RNA (e.g., a U6c promoter), a nucleotide sequence encoding a second guide RNA, and a nucleotide sequence encoding a second guide RNA scaffold. In some embodiments, the second vector comprises a staffer sequence (e.g., a 3’UTR desmin sequence) between the reverse complement of the promoter for expression of the first guide RNA and the promoter for expression of the second guide RNA. In some embodiments, the second vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a first copy of a first guide RNA scaffold, the reverse complement of a nucleotide sequence encoding a first copy of the first guide RNA, the reverse complement of a promoter for expression of the first copy of the first guide RNA (e.g., a 7SK2 promoter), the reverse complement of a second copy of the nucleotide sequence encoding the first guide RNA scaffold, the reverse complement of a second copy of the nucleotide sequence encoding the first guide RNA, the reverse complement of a promoter for expression of the second copy of the nucleotide sequence encoding the first guide RNA (e.g., a hU6c promoter), a promoter for expression of a first copy of a second guide RNA (e.g., a hU6c promoter), a first copy of a nucleotide sequence encoding a second guide RNA, a first copy of a nucleotide sequence encoding a second guide RNA scaffold, a promoter for expression of a second copy of the second guide RNA (e.g., a 7Sk2 promoter), a second copy of the nucleotide sequence encoding the second guide RNA, and a second copy of the nucleotide sequence encoding the second guide RNA scaffold. In some embodiments, the second vector comprises a staffer sequence (e.g., a 3’UTR desmin sequence) between the reverse complement of the promoter for expression of the second copy of the first guide RNA and the promoter for expression of the first copy of the second guide RNA. In some embodiments, the second vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a first copy of a first guide RNA scaffold, the reverse complement of a first copy of a nucleotide sequence encoding the first guide RNA, the reverse complement of a promoter for expression of the first copy of the first guide RNA (e.g., a 7SK2 promoter), the reverse complement of a first copy of a nucleotide sequence encoding a second guide RNA scaffold, the reverse complement of a nucleotide sequence encoding the first copy of the second guide RNA, the reverse complement of a promoter for expression of the first copy of the second guide RNA (e.g., a hU6c promoter), a promoter for expression of a second copy of the second guide RNA (e.g., a hU6c promoter), a second copy of the nucleotide sequence encoding the second guide RNA, a second copy of the nucleotide sequence encoding the second guide RNA scaffold, a promoter for expression of a second copy of the first guide RNA (e.g., a 7SK2 promoter), a second copy of the nucleotide sequence encoding the first guide RNA, and a second copy of the nucleotide sequence encoding the first guide RNA scaffold. In some embodiments, the second vector comprises a staffer sequence (e.g., a 3’UTR desmin sequence) between the reverse complement of the promoter for expression of the first copy of the second guide RNA and the promoter for expression of the second copy of the first guide RNA. In some embodiments, the second vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a nucleotide sequence encoding a first guide RNA scaffold, the reverse complement of a nucleotide sequence encoding a first guide RNA, the reverse complement of a promoter for expression of a first guide RNA (e.g., a hU6c promoter), a promoter for expression of a second guide RNA (e.g., a hU6c promoter), a nucleotide sequence encoding a second guide RNA, and a nucleotide sequence encoding a second guide RNA scaffold. In some embodiments, the second vector comprises a staffer sequence (e.g., a 3’UTR desmin sequence) between the reverse complement of the promoter for expression of the first guide RNA and the promoter for expression of the second guide RNA. In particular embodiments, the first guide RNA is different from the second guide RNA. In some embodiments, the first guide RNA comprises a sequence of Tables 1A-1C and the second guide RNA comprises a different sequence from Tables 1A-1C. In some embodiments, the first guide RNA comprises a sequence of Table 1A and the second guide RNA comprises a sequence from Table 1C. In some embodiments, the first guide RNA comprises a sequence of Table 1A and the second guide RNA comprises a sequence from Table 1B. In some embodiments, the first guide RNA comprises a sequence of Table 1B and the second guide RNA comprises a sequence from Table 1C.

[00205] In some embodiments, if the composition comprises one or more nucleic acids encoding an RNA-targeted endonuclease and one or more guide RNAs, the one or more nucleic acids are designed such that they express the one or more guide RNAs at an equivalent or higher level (e.g., a greater number of expressed transgene copies) as compared to the expression level of the RNA- targeted endonuclease. In some embodiments, the one or more nucleic acids are designed such that they express (e.g., on average in 100 cells) the one or more guide RNAs at least a 1.1, 1.2, 1.3, 1.4, or 1.5 times higher level (e.g., a greater number of expressed transgene copies) as compared to the expression level of the RNA-targeted endonuclease. In some embodiments, the one or more nucleic acids are designed such that they express the one or more guide RNAs at 1.01-1.5, 1.01-1.4, 1.01-1.3, 1.01-1.2, 1.01-1.1, 1.1-2.0, 1.1-1.8, 1.1-1.6, 1.1-1.4, 1.1-1.3, 1.2-2.0, 1.2-1.8, 1.2-1.6, 1.2-1.4, 1.4-2.0, 1.4-1.8, 1.4-1.6, 1.6-2.0, 1.6-1.8, or 1.8-2.0 times higher level (e.g., a greater number of expressed transgene copies) as compared to the expression level of the RNA-targeted endonuclease. In some embodiments, the one or more guide RNAs are designed to express a higher level than the RNA- targeted endonuclease by: a) utilizing one or more regulatory elements (e.g., promoters or enhancers) that express the one or more guide RNAs at a higher level as compared to the regulatory elements (e.g., promoters or enhancers) for expression of the RNA-targeted endonuclease; and/or b) expressing more copies of one or more of the guide RNAs as compared to the number of copies of the RNA- targeted endonuclease (e.g., 2x or 3x as many copies of the nucleotide sequences encoding the one or more guide RNAs as compared to the number of copies of the nucleotide sequences encoding the RNA-targeted endonuclease). For example, in some embodiments, the composition comprises multiple nucleic acid molecules (e.g., in multiple vectors), wherein for every nucleotide sequence encoding an RNA-targeted endonuclease in the nucleic acid molecules in the composition, there are two or three copies of the nucleotide sequence encoding the guide RNA in the nucleic acid molecules in the composition. In some embodiments, the composition comprises a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA are not the same (e.g., any of the guide RNA pairs disclosed herein), and for every nucleotide sequence encoding an RNA- targeted endonuclease in the nucleic acid molecules in the composition, there are two or three copies of the nucleotide sequence encoding the first guide RNA and/or the second guide RNA.

Endonucleases

[00206] In some embodiments, any of the nucleic acids disclosed herein encodes an RNA-targeted endonuclease. In some embodiments, the RNA-targeted endonuclease has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA- targeted endonuclease comprises a Cas nuclease. Examples of Cas9 nucleases include those of the type II CRISPR systems. [00207] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 941 (designated herein as SpCas9):

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT

RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL VQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA

PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNE KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE

MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK MKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM

NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE

VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTL TNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

[00208] In some embodiments, the nucleic acid encoding SaCas9 encodes an SaCas9 comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 711:

KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI

QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE DTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK

AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY RVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEE IEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDD FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIR

TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL VKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ

KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYK

HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHI

KDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP

EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN KLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC YEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN DKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[00209] In some embodiments, the nucleic acid encoding SaCas9 comprises the nucleic acid of

SEQ ID NO: 9014:

[00210] A AGCGC A ATT AC ATCCTGGGCCTGG AT ATCGGC ATC ACCTCCGTGGGCT ACG

GCATCATCGACTATGAGACACGGGATGTGATCGACGCCGGCGTGAGACTGTTCAAGGAG

GCCAACGTGGAGAACAATGAGGGCCGGCGGAGCAAGAGGGGAGCAAGGCGCCTGAAGC

GGAGAAGGCGCCACAGAATCCAGAGAGTGAAGAAGCTGCTGTTCGATTACAACCTGCTG

ACCGACCACTCCGAGCTGTCTGGCATCAATCCTTATGAGGCCCGGGTGAAGGGCCTGTCC

CAGAAGCTGTCTGAGGAGGAGTTTTCTGCCGCCCTGCTGCACCTGGCAAAGAGGAGAGG

CGTGCACAACGTGAATGAGGTGGAGGAGGACACCGGCAACGAGCTGAGCACAAAGGAG

CAGATCAGCCGCAATTCCAAGGCCCTGGAGGAGAAGTATGTGGCCGAGCTGCAGCTGGA

GCGGCTGAAGAAGGATGGCGAGGTGAGGGGCTCCATCAATCGCTTCAAGACCTCTGACT

ACGTGAAGGAGGCCAAGCAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGATCAG

AGCTTTATCGATACATATATCGACCTGCTGGAGACCAGGCGCACATACTATGAGGGACC

AGGAGAGGGCTCCCCCTTCGGCTGGAAGGACATCAAGGAGTGGTACGAGATGCTGATGG

GCCACTGCACCTATTTTCCAGAGGAGCTGAGATCCGTGAAGTACGCCTATAACGCCGATC

TGTACAACGCCCTGAATGACCTGAACAACCTGGTCATCACCAGGGATGAGAACGAGAAG

CTGGAGTACTATGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCAGAAGAAGAAGCC

TACACTGAAGCAGATCGCCAAGGAGATCCTGGTGAACGAGGAGGACATCAAGGGCTACC

GCGTGACCAGCACAGGCAAGCCAGAGTTCACCAATCTGAAGGTGTATCACGATATCAAG

GACATCACAGCCCGGAAGGAGATCATCGAGAACGCCGAGCTGCTGGATCAGATCGCCAA

GATCCTGACCATCTATCAGAGCTCCGAGGACATCCAGGAGGAGCTGACCAACCTGAATA

GCGAGCTGACACAGGAGGAGATCGAGCAGATCAGCAATCTGAAGGGCTACACCGGCAC

ACACAACCTGTCCCTGAAGGCCATCAATCTGATCCTGGATGAGCTGTGGCACACAAACG

ACAATCAGATCGCCATCTTTAACAGGCTGAAGCTGGTGCCAAAGAAGGTGGACCTGAGC

CAGCAGAAGGAGATCCCAACCACACTGGTGGACGATTTCATCCTGTCCCCCGTGGTGAA

GCGGAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGC

CCAATGATATCATCATCGAGCTGGCCAGGGAGAAGAACTCTAAGGACGCCCAGAAGATG

ATCAATGAGATGCAGAAGAGGAACCGCCAGACCAATGAGCGGATCGAGGAGATCATCA

GAACCACAGGCAAGGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGATAT GCAGGAGGGCAAGTGTCTGTATAGCCTGGAGGCCATCCCTCTGGAGGACCTGCTGAACA ATCCATTCAACTACGAGGTGGATCACATCATCCCCCGGAGCGTGAGCTTCGACAATTCCT TTAACAATAAGGTGCTGGTGAAGCAGGAGGAGAACTCTAAGAAGGGCAATAGGACCCCT TTCCAGTACCTGTCTAGCTCCGATTCTAAGATCAGCTACGAGACCTTCAAGAAGCACATC CTGAATCTGGCCAAGGGCAAGGGCCGCATCTCTAAGACCAAGAAGGAGTACCTGCTGGA GGAGCGGGACATCAACAGATTCAGCGTGCAGAAGGACTTCATCAACCGGAATCTGGTGG ACACCAGATACGCCACACGCGGCCTGATGAATCTGCTGCGGTCCTATTTCAGAGTGAACA ATCTGGATGTGAAGGTGAAGAGCATCAACGGCGGCTTCACCTCCTTTCTGCGGAGAAAG TGGAAGTTTAAGAAGGAGAGAAACAAGGGCTATAAGCACCACGCCGAGGATGCCCTGAT CATCGCCAATGCCGACTTCATCTTTAAGGAGTGGAAGAAGCTGGACAAGGCCAAGAAAG

TGATGGAGAACCAGATGTTCGAGGAGAAGCAGGCCGAGAGCATGCCCGAGATCGAGAC CGAGCAGGAGTACAAGGAGATTTTCATCACACCTCACCAGATCAAGCACATCAAGGACT TCAAGGACTACAAGTATTCCCACAGGGTGGATAAGAAGCCCAACCGCGAGCTGATCAAT GACACCCTGTATTCTACAAGGAAGGACGATAAGGGCAATACCCTGATCGTGAACAATCT GAACGGCCTGTACGACAAGGATAATGACAAGCTGAAGAAGCTGATCAACAAGAGCCCC GAGAAGCTGCTGATGTACCACCACGATCCTCAGACATATCAGAAGCTGAAGCTGATCAT GGAGCAGTACGGCGACGAGAAGAACCCACTGTATAAGTACTATGAGGAGACCGGCAACT ACCTGACAAAGTATTCCAAGAAGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTAT GGCAACAAGCTGAATGCCCACCTGGACATCACCGACGATTACCCCAACAGCCGGAATAA GGTGGTGAAGCTGAGCCTGAAGCCATACAGGTTCGACGTGTACCTGGACAACGGCGTGT ATAAGTTTGTGACAGTGAAGAATCTGGATGTGATCAAGAAGGAGAACTACTATGAAGTG AATAGCAAGTGCTACGAGGAGGCCAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGT TCATCGCCTCTTTTTACAACAATGACCTGATCAAGATCAATGGCGAGCTGTATAGAGTGA TCGGCGTGAACAATGATCTGCTGAACCGCATCGAAGTGAATATGATCGACATCACCTACC GGGAGTATCTGGAGAACATGAATGATAAGAGGCCCCCTCGCATCATCAAGACCATCGCC TCTAAGACACAGAGCATCAAGAAGTACTCTACAGACATCCTGGGCAACCTGTATGAGGT

GAAGAGCAAGAAGCACCCTCAGATCATCAAGAAGGGC.In some embodiments comprising a nucleic acid encoding SaCas9, the SaCas9 comprises an amino acid sequence of SEQ ID NO: 711.

[00211] In some embodiments, the SaCas9 is a variant of the amino acid sequence of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an E at the position corresponding to position 781 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an N at the position corresponding to position 967 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 967 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an H at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an E at the position corresponding to position 781 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 967 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 711; a K at the position corresponding to position 967 of SEQ ID NO: 711; and an H at the position corresponding to position 1014 of SEQ ID NO: 711.

[00212] In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an N at the position corresponding to position 412 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an N at the position corresponding to position 418 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 412 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 418 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 244 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 412 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 418 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 244 of SEQ ID NO: 711; an A at the position corresponding to position 412 of SEQ ID NO: 711; an A at the position corresponding to position 418 of SEQ ID NO: 711; and an A at the position corresponding to position 653 of SEQ ID NO: 711.

[00213] In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 412 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 418 of SEQ ID NO: 711; an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711; an amino acid other than an E at the position corresponding to position 781 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 967 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 244 of SEQ ID NO: 711; an A at the position corresponding to position 412 of SEQ ID NO: 711; an A at the position corresponding to position 418 of SEQ ID NO: 711; an A at the position corresponding to position 653 of SEQ ID NO: 711; a K at the position corresponding to position 781 of SEQ ID NO: 711; a K at the position corresponding to position 967 of SEQ ID NO: 711; and an H at the position corresponding to position 1014 of SEQ ID NO: 711.

[00214] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 715 (designated herein as SaCas9-KKH or SACAS9KKH):

KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE DTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK

AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY RVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEE

IEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDD FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIR TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL

VKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYK HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHI

KDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN KLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC

YEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN DKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[00215] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 716 (designated herein as SaCas9-HF):

KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE DTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK

AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELASVKYA YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY RVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEE

IEQISNLKGYTGTHNLSLKAINLILDELWHTNDAQIAIFARLKLVPKKVDLSQQKEIPTTLVDD FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIR TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL

VKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ KDFINRNLVDTRYATAGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYK HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHI KDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN

KLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC

YEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN DKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[00216] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 717 (designated herein as SaCas9-KKH-HF):

KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI

QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE

DTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELASVKYA YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY

RVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEE

IEQISNLKGYTGTHNLSLKAINLILDELWHTNDAQIAIFARLKLVPKKVDLSQQKEIPTTLVDD

FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIR

TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL

VKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ

KDFINRNLVDTRYATAGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYK

HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHI

KDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP

EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN KLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC YEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN DKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[00217] In some embodiments, the nucleic acid encoding SluCas9 encodes a SluCas9 comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 712:

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE

RVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDD

VGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH

QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYAY

SADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI

TKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDK ENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEF ILSPVVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIG KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV LVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE VQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQ DIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK LGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDK LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP RIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00218] In some embodiments, the SluCas9 is a variant of the amino acid sequence of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises a K at the position corresponding to position 966 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an H at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712; and an amino acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO: 712; and an H at the position corresponding to position 1013 of SEQ ID NO: 712.

[00219] In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an N at the position corresponding to position 414 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than a T at the position corresponding to position 420 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712; an amino acid other than an N at the position corresponding to position 414 of SEQ ID NO: 712; an amino acid other than a T at the position corresponding to position 420 of SEQ ID NO: 712; and an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 414 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 420 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712; an A at the position corresponding to position 414 of SEQ ID NO: 712; an A at the position corresponding to position 420 of SEQ ID NO: 712; and an A at the position corresponding to position 655 of SEQ ID NO: 712.

[00220] In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712; an amino acid other than an N at the position corresponding to position 414 of SEQ ID NO: 712; an amino acid other than a T at the position corresponding to position 420 of SEQ ID NO: 712; an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712; an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO: 712; and an amino acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712; an A at the position corresponding to position 414 of SEQ ID NO: 712; an A at the position corresponding to position 420 of SEQ ID NO: 712; an A at the position corresponding to position 655 of SEQ ID NO: 712; a K at the position corresponding to position 781 of SEQ ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO: 712; and an H at the position corresponding to position 1013 of SEQ ID NO: 712.

[00221] In some embodiments, the SluCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 718 (designated herein as SluCas9-KH or SLUCAS9KH):

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE RVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDD VGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYAY SADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI TKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDK ENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEF ILSPVVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIG KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV LVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE VQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQ DIKDFRNFKYSHRVDKKPNRKLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK LGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDK

LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP HIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN. [00222] In some embodiments, the SluCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 719 (designated herein as SluCas9-HF):

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE RVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDD VGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH

QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELASVKYAY SADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI TKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDK

ENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRAQMEIFAHLNIKPKKINLTAANKIPKAMIDEF ILSPVVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIG KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV

LVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE VQKEFINRNLVDTRYATAELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQ

DIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK LGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDK

LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP RIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00223] In some embodiments, the SluCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 720 (designated herein as SluCas9-HF-KH):

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE RVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDD VGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH

QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELASVKYAY SADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI TKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDK

ENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRAQMEIFAHLNIKPKKINLTAANKIPKAMIDEF ILSPVVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIG KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV

LVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE VQKEFINRNLVDTRYATAELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQ

DIKDFRNFKYSHRVDKKPNRKLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK LGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDK LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP HIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00224] In some embodiments, the Cas protein is any of the engineered Cas proteins disclosed in

Schmidt et al., 2021, Nature Communications, “Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.”

[00225] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7021 (designated herein as sRGNl):

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL

DRVKHLLAEYDLLDLTNIPKSTNPYQTRVKGLNEKLSKDELVIALLHIAKRRGIHNVDVAAD

KEETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDT

QMQYYPEIDETFKEKYISLVETRREYFEGPGKGSPFGWEGNIKKWFEQMMGHCTYFPEELRS

VKYSYSAELFNALNDLNNLVITRDEDAKLNYGEKFQIIENVFKQKKTPNLKQIAIEIGVHETEI KGYRVNKSGTPEFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIYQDEQSIKEELNKLPEILN EQDKAEIAKLIGYNGTHRLSLKCIHLINEELWQTSRNQMEIFNYLNIKPNKVDLSEQNKIPKD MVNDFILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRI

NEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLKDIPLEDLLRNPNNYDIDHIIPRSVSFDDSM

HNKVLVRREQNAKKNNQTPYQYLTSGYADIKYSVFKQHVLNLAENKDRMTKKKREYLLEE RDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKF KKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFI IPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQ

FDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLK YIGNKLGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPE QKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELN NIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00226] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7022 (designated herein as sRGN2):

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL

ERVKSLLSEYKIISGLAPTNNQPYNIRVKGLTEQLTKDELAVALLHIAKRRGIHKIDVIDSNDD VGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH QLDENFINKYIELVEMRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYA YSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYR ITKSGTPEFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDK ENIAQLTGYNGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEF ILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQ TGNQNAKRIVEKIRLHDQQEGKCLYSLESIALMDLLNNPQNYEVDHIIPRSVAFDNSIHNKVL VKQIENSKKGNRTPYQYLNSSDAKLSYNQFKQHILNLSKSKDRISKKKKDYLLEERDINKFEV QKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRNHGY KHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQDIK DFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPEKFL MYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGS HLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDKLKL GKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEPRIK KTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00227] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7023 (designated herein as sRGN3):

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE ETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQM QYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLM SEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIP TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER DINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFK KERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFII

PKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQF DKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLK YIGNKLGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPE QKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELN NIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00228] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7024 (designated herein as sRGN3.1):

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE ETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQM QYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLM SEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIP TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER DINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFK KERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFII PKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQF DKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLK YIGNKLGSHLDVTHQFKSSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIP KDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEI NNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[00229] In some embodiments, the Cas9 comprises an amino acid sequence that is encoded by a nucleic acid molecule at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 918 (an exemplary nucleic acid molecule encoding SRGN3.1):

ATGAATCAGAAATTCATCCTGGGACTGGACATCGGCATTACCTCTGTGGGCTACGGCTTG

ATTGACTACGAGACCAAGAACATCATCGACGCCGGCGTTAGACTGTTTCCTGAGGCCAAT

GTGGAAAACAACGAGGGCAGACGGTCTAAGCGGGGCTCTAGACGACTGAAAAGAAGAA

GAATCCACAGACTGGAAAGAGTGAAGCTGCTGCTGACCGAGTACGACCTGATCAATAAG

GAACAGATCCCTACAAGCAACAACCCCTACCAGATTAGAGTGAAGGGCCTGAGCGAGAT

CCTGAGCAAAGACGAGCTGGCCATCGCCCTGCTGCACCTGGCCAAGCGGAGGGGCATCC

ACAACGTGGATGTGGCCGCCGACAAGGAGGAAACCGCCAGCGACTCCCTGAGCACCAAA

GATCAGATCAACAAGAATGCCAAGTTCCTGGAGAGCAGATACGTGTGCGAGCTGCAGAA

GGAGAGATTGGAGAACGAGGGCCACGTGCGGGGCGTGGAGAATAGATTCCTGACAAAA

GATATCGTGCGGGAAGCCAAGAAGATCATCGACACCCAGATGCAGTACTATCCTGAAAT

CGACGAAACCTTCAAGGAGAAGTACATCAGCCTGGTCGAAACCAGACGCGAGTATTTCG

AGGGCCCCGGACAGGGCTCTCCTTTCGGCTGGAACGGCGATCTGAAGAAGTGGTACGAG

ATGCTGATGGGACACTGCACCTACTTCCCTCAAGAGCTGAGATCTGTGAAGTACGCCTAC

AGCGCCGATCTGTTCAACGCCCTGAACGATCTGAACAACCTTATCATCCAGCGGGACAAT

TCTGAGAAGCTGGAATACCACGAGAAATATCATATCATCGAGAACGTCTTTAAACAAAA

GAAAAAGCCTACCCTGAAGCAGATCGCCAAGGAGATCGGAGTGAATCCTGAGGATATCA

AAGGCTACAGAATCACAAAGTCCGGCACCCCTGAGTTCACCAGCTTTAAGCTGTTCCACG

ACCTGAAGAAGGTCGTGAAAGACCACGCCATCCTCGATGACATCGATCTGCTGAACCAG

ATCGCTGAGATCCTGACAATCTACCAGGACAAAGATTCTATCGTGGCTGAACTGGGACA GCTGGAATACCTTATGAGCGAGGCCGACAAGCAAAGCATCTCCGAACTGACCGGCTACA CGGGCACCCATAGCCTGAGCCTGAAGTGTATGAACATGATCATCGATGAGCTGTGGCAC TCTAGCATGAACCAGATGGAAGTTTTCACCTACCTGAACATGCGGCCTAAGAAGTACGA GCTGAAGGGCTACCAGAGAATCCCCACCGATATGATCGACGACGCCATCCTGAGCCCCG TGGTGAAAAGAACATTCATCCAGAGCATCAACGTGATCAACAAGGTGATCGAGAAGTAC

GGCATTCCAGAGGACATCATCATCGAGCTGGCCAGAGAAAACAACAGCGACGATAGAA AGAAGTTTATCAACAACCTGCAGAAAAAAAACGAGGCCACCCGGAAGAGAATTAATGA GATCATCGGCCAGACAGGCAACCAGAACGCCAAAAGGATCGTGGAAAAAATCAGACTG

CACGACCAGCAGGAGGGCAAGTGCCTGTACTCTCTGGAAAGCATCCCCCTGGAGGACCT GCTGAACAATCCAAATCACTACGAGGTGGACCACATCATCCCTAGAAGCGTCAGCTTCG ACAACAGCTACCACAACAAGGTGCTGGTGAAGCAGAGCGAAAACAGTAAGAAATCCAA

CCTGACACCTTACCAGTACTTTAACAGCGGCAAGAGCAAGCTGAGCTACAACCAGTTCA AGCAGCACATCCTGAACCTGTCCAAATCTCAGGATAGAATCTCCAAGAAAAAGAAGGAA TACCTGCTGGAGGAGAGAGATATCAACAAGTTCGAAGTCCAAAAGGAGTTCATCAACAG

GAACCTGGTGGACACCCGGTACGCCACAAGAGAGCTGACAAACTACCTGAAAGCCTACT TCAGCGCTAACAACATGAACGTGAAGGTGAAAACCATCAATGGAAGTTTCACAGACTAC CTTCGGAAGGTGTGGAAGTTCAAGAAGGAACGGAATCACGGCTACAAGCACCACGCAGA

GGACGCCCTGATTATAGCTAATGCCGATTTCCTGTTCAAAGAAAACAAGAAGCTGAAAG CCGTGAACAGCGTTCTGGAAAAGCCTGAAATTGAGACCAAGCAACTGGATATACAGGTG GACAGCGAGGACAACTACTCCGAGATGTTCATCATCCCTAAACAGGTGCAGGACATCAA

AGACTTCAGAAATTTCAAGTACAGCCACAGAGTGGACAAGAAACCCAACCGGCAGCTGA

TCAATGACACCCTGTATAGCACCCGCAAGAAGGATAACAGCACCTACATCGTCCAGACC ATCAAGGACATCTACGCTAAGGACAACACCACCCTGAAAAAGCAATTTGATAAGTCCCC CGAAAAGTTTCTGATGTACCAACACGATCCTAGAACCTTCGAAAAGTTGGAGGTGATCAT

GAAGCAATATGCCAACGAGAAGAACCCACTGGCCAAGTACCATGAGGAGACAGGAGAA TACCTGACCAAGTATTCTAAGAAGAATAACGGCCCCATCGTGAAGTCCCTGAAGTACATT GGGAACAAACTCGGAAGCCACCTGGACGTGACGCACCAGTTCAAGAGCAGCACCAAGA

AGCTAGTGAAACTGAGCATCAAGAACTACAGATTCGACGTGTACCTGACAGAAAAGGGC TACAAATTTGTGACCATCGCTTACCTGAATGTGTTCAAAAAGGACAATTATTACTACATC CCAAAGGACAAGTACCAGGAGCTTAAAGAGAAAAAAAAGATCAAGGATACCGACCAGT

TTATCGCTAGCTTCTACAAGAACGACCTGATTAAGCTGAACGGCGACCTGTACAAGATCA

TCGGCGTGAACTCTGACGACCGGAACATAATAGAGCTGGATTATTATGACATCAAGTAC AAGGACTACTGCGAGATCAACAACATCAAGGGCGAGCCTAGAATCAAAAAGACCATCG GGAAGAAAACCGAGTCTATCGAAAAGTTTACAACAGACGTGCTGGGCAACCTGTACCTG

CACAGCACGGAAAAGGCCCCTCAGCTCATCTTCAAGAGAGGCCTG

[00230] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7025 (designated herein as sRGN3.2): MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE ETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQM QYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLM SEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIP TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER DINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFK KERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFII PKQVQDIKDFRNFKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKKQ FNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIK LLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYI PKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYC EINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[00231] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7026 (designated herein as sRGN3.3):

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE ETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQM QYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLM SEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIP TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER DINKFEVQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFD KYRNHGYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKEEDYNNVFE TPKLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKK QFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKI

KLLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYY IPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYC EINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL. [00232] In some embodiments, the Cas9 comprises an amino acid sequence that is encoded by a nucleic acid molecule at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 919 (an exemplary nucleic acid molecule encoding SRGN3.3):

AACCAGAAGTTTATCCTGGGCCTGGATATCGGCATCACATCCGTGGGCTACGGCCTGATT GATTACGAAACCAAGAACATCATTGATGCCGGCGTCCGGCTTTTCCCTGAAGCTAACGTG GAAAACAATGAGGGCAGACGGAGCAAGAGAGGCAGCAGACGGCTGAAGCGGAGAAGA ATCCATAGACTCGAACGGGTGAAGCTGCTGCTGACCGAGTACGACCTGATCAACAAGGA

GCAGATCCCCACCAGCAACAACCCATACCAGATCAGAGTGAAAGGCCTTTCTGAGATTC TGAGCAAGGATGAGCTGGCTATCGCTCTGCTCCACCTGGCCAAGAGAAGGGGAATCCAC AACGTTGACGTGGCCGCCGATAAGGAAGAGACCGCCAGCGATAGCCTGAGCACCAAGG ACCAGATCAACAAGAACGCCAAGTTCCTGGAAAGCAGATACGTGTGCGAGCTGCAAAAA

GAGAGACTGGAAAATGAGGGCCATGTGCGGGGCGTTGAGAACAGATTCCTGACCAAAG ACATCGTCAGAGAGGCCAAGAAAATTATTGACACCCAGATGCAGTACTATCCTGAGATA GACGAGACCTTTAAGGAAAAGTACATCAGCCTGGTGGAAACAAGAAGAGAATACTTCGA AGGACCTGGCCAGGGCTCCCCTTTCGGCTGGAACGGCGACCTGAAGAAGTGGTACGAGA

TGCTGATGGGCCACTGCACCTACTTCCCCCAGGAGCTGCGGAGCGTGAAGTACGCTTACA GCGCCGACCTGTTCAATGCCCTGAACGACCTGAACAATCTTATCATCCAAAGAGATAACA GCGAGAAATTAGAATACCACGAGAAGTACCACATCATCGAGAATGTGTTCAAGCAAAAG AAGAAGCCTACCCTGAAGCAGATCGCCAAAGAGATCGGCGTGAACCCTGAGGACATCAA

GGGCTATCGGATCACCAAGTCCGGCACCCCTGAATTCACCAGCTTCAAGCTGTTTCACGA CCTCAAAAAGGTCGTGAAGGACCACGCCATCCTGGACGACATCGATCTGCTGAATCAGA TCGCCGAGATCCTGACCATCTACCAGGACAAGGACTCTATCGTGGCCGAGCTTGGACAG CTGGAGTACCTGATGAGCGAGGCTGACAAGCAGAGCATCAGCGAGCTGACCGGCTACAC

CGGAACCCACAGCCTGTCCCTGAAGTGCATGAACATGATCATCGACGAGCTGTGGCACT CTAGCATGAACCAGATGGAAGTGTTCACCTACCTGAACATGAGACCTAAGAAGTACGAA CTGAAGGGTTACCAGAGAATCCCAACCGACATGATCGACGACGCCATCCTGAGCCCCGT GGTGAAGCGGACCTTTATCCAGAGCATCAATGTGATCAACAAGGTGATCGAGAAATACG

GCATCCCCGAGGACATCATCATCGAACTGGCCAGAGAGAATAACTCTGATGACCGGAAG AAGTTCATCAACAACCTGCAGAAGAAGAACGAAGCCACCAGAAAGCGCATCAACGAGA TCATCGGCCAAACAGGGAATCAGAACGCCAAGAGGATCGTGGAAAAGATTCGGCTGCAC GACCAGCAGGAGGGAAAATGCCTGTACAGCCTGGAAAGCATCCCCCTGGAAGATCTACT

GAACAACCCCAACCACTACGAGGTGGATCACATCATCCCTAGAAGCGTGTCCTTTGACA ACAGTTACCACAACAAGGTGCTGGTGAAGCAATCCGAGAACTCGAAGAAGAGCAACCTG ACACCTTACCAGTACTTTAACAGCGGCAAGTCCAAGCTGTCTTATAACCAGTTCAAGCAG CACATCCTCAACCTGTCAAAGTCTCAGGACAGAATCTCTAAGAAGAAGAAGGAGTATCT

GCTGGAAGAGCGGGACATCAACAAGTTCGAGGTGCAGAAAGAGTTCATTAACAGAAACC TGGTCGACACCCGGTACGCCACACGCGAACTGACAAGCTACCTGAAGGCCTACTTCTCCG CCAACAATATGGACGTGAAGGTCAAGACCATCAATGGCAGCTTCACAAATCACCTGAGA AAGGTCTGGCGGTTCGACAAGTACAGAAACCACGGCTACAAGCACCACGCCGAGGATGC TCTGATTATCGCCAACGCCGACTTCCTGTTCAAGGAAAACAAAAAACTGCAGAACACCA ACAAGATCCTGGAAAAACCTACAATCGAGAACAACACAAAGAAAGTGACAGTGGAAAA AGAGGAAGATTACAACAACGTGTTTGAGACACCTAAGCTGGTTGAGGATATCAAGCAGT ACCGGGACTATAAGTTTAGCCACAGAGTGGACAAGAAACCTAACAGGCAGCTGATCAAC GACACTCTGTACAGCACAAGAATGAAAGATGAGCACGATTACATCGTGCAGACCATTAC CGACATCTACGGCAAGGACAACACCAATCTGAAGAAGCAGTTCAACAAAAATCCCGAGA AGTTCCTGATGTACCAAAATGACCCTAAGACATTCGAGAAGCTGAGCATCATCATGAAA CAGTACTCTGATGAGAAGAACCCACTGGCCAAGTACTACGAGGAAACAGGCGAATACCT GACCAAGTACTCTAAGAAGAACAACGGCCCTATCGTGAAGAAGATCAAACTGCTGGGCA ACAAAGTGGGAAATCATCTGGATGTGACCAATAAATACGAGAACTCTACAAAGAAACTG GTGAAGCTGAGCATTAAGAACTACAGATTCGACGTCTACCTGACAGAAAAGGGATACAA GTTCGTGACCATCGCCTACCTGAACGTGTTCAAGAAAGACAACTACTACTACATCCCAAA AGACAAGTACCAAGAGTTAAAAGAGAAGAAGAAGATAAAGGATACCGACCAGTTTATC GCTTCTTTCTACAAGAACGACCTGATCAAGCTGAACGGTGATCTGTACAAAATCATCGGA GTGAATAGCGATGACAGAAATATCATCGAACTGGATTACTATGACATCAAGTACAAAGA TTATTGTGAAATCAACAACATCAAGGGAGAGCCCAGAATTAAGAAAACCATCGGCAAGA AAACAGAGAGCATCGAGAAATTCACCACAGATGTGCTGGGCAACCTGTACCTGCACAGC ACAGAGAAAGCCCCTCAGCTCATCTTCAAGAGAGGCCTG

[00233] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7027 (designated herein as sRGN4):

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHNINVSSEDE DASNELSTKEQINRNNKLLKDKYVCEVQLQRLKEGQIRGEKNRFKTTDILKEIDQLLKVQKD YHNLDIDFINQYKEIVETRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKY AYSADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKG YRITKSGKPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLMS EADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPT DMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKR INEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSY HNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDI

NKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKE RNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPK QVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFD KSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYI GNKLGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQK YDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNI KGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[00234] In some embodiments, the Cas9 comprises an amino acid sequence that is encoded by a nucleic acid molecule at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 920 (an exemplary nucleic acid molecule encoding sRGN4):

ATGAATCAAAAGTTTATCCTGGGTCTGGACATCGGCATCACATCTGTGGGCTACGGCCTT ATCGATTACGAGACCAAGAATATCATTGATGCTGGCGTACGGCTGTTCCCTGAGGCTAAC GTGGAAAACAACGAGGGTAGACGGAGCAAGAGAGGCAGCAGACGGCTGAAACGGCGTA GAATCCACCGGCTGGAGAGAGTGAAGAAGTTGCTGGAAGATTACAACTTGCTGGATCAA TCCCAGATCCCCCAGAGCACTAATCCTTATGCTATCCGGGTGAAGGGCCTGTCTGAAGCC CTGAGCAAAGACGAGCTGGTGATTGCCCTGCTGCACATCGCGAAGAGAAGAGGCATCCA CAATATCAATGTGTCCTCTGAGGATGAGGATGCCAGCAACGAGCTGAGCACTAAGGAAC AGATCAATCGGAACAACAAGCTGCTGAAGGACAAGTACGTGTGTGAAGTGCAGCTGCAG AGACTGAAGGAAGGCCAGATAAGAGGCGAAAAGAACAGATTCAAGACAACAGACATCC TGAAAGAAATCGACCAGCTGCTGAAGGTCCAGAAGGACTACCACAACCTCGACATCGAT TTCATTAACCAGTACAAGGAAATCGTGGAAACCAGGAGAGAGTACTTCGAGGGCCCTGG CAAGGGCTCCCCATACGGCTGGGAGGGCGACCCCAAGGCCTGGTACGAAACCCTGATGG GCCACTGCACCTACTTCCCCGACGAACTGAGAAGCGTCAAGTACGCCTACAGCGCCGAT CTGTTCAACGCCCTGAACGACCTGAACAACCTGGTGATCCAGCGGGACGGCCTGAGCAA ACTGGAGTACCATGAAAAGTATCATATCATCGAGAACGTGTTCAAGCAGAAGAAAAAAC CTACCCTGAAGCAGATCGCCAACGAGATCAACGTGAACCCTGAGGACATTAAAGGCTAC AGAATCACCAAAAGCGGCAAGCCAGAGTTCACCAGCTTCAAGCTGTTTCACGACCTGAA GAAGGTCGTGAAAGACCACGCCATCCTGGACGACATCGATCTGCTTAACCAGATCGCTG AAATCCTCACAATCTACCAGGACAAGGACTCTATCGTGGCCGAGCTGGGACAGCTGGAA TACCTGATGAGCGAGGCCGATAAGCAGAGCATCAGCGAGCTGACCGGCTACACCGGAAC CCACAGCCTGAGCCTGAAGTGTATGAACATGATCATCGACGAGCTGTGGCACAGCTCTAT GAACCAGATGGAAGTATTCACCTACCTGAACATGAGACCTAAGAAGTACGAACTGAAGG GCTATCAGAGAATCCCTACAGACATGATCGACGATGCCATCCTGTCTCCTGTGGTGAAGA GAACCTTCATCCAGTCTATCAACGTGATCAACAAGGTGATCGAAAAGTACGGAATCCCT GAAGATATCATCATCGAACTGGCCAGAGAGAACAACTCCGACGACAGAAAGAAATTCAT

CAACAACCTGCAGAAGAAGAATGAGGCCACACGGAAGCGGATTAATGAGATCATCGGC CAAACCGGCAACCAGAACGCCAAAAGAATCGTGGAAAAGATCCGGCTGCACGATCAGC AGGAGGGCAAATGCCTGTACAGCCTGGAGAGCATCCCCCTGGAGGACCTGCTCAACAAC CCCAACCACTACGAGGTGGATCACATCATCCCAAGATCTGTTAGCTTCGACAACAGCTAC CACAACAAGGTGCTGGTGAAGCAAAGCGAAAACTCTAAGAAATCTAACCTGACACCTTA CCAGTACTTTAACAGCGGCAAGTCCAAGCTGTCTTATAACCAGTTTAAGCAGCACATCCT GAACCTGAGCAAGTCCCAGGATAGAATCAGCAAAAAAAAGAAGGAATACCTGCTGGAG

GAACGCGACATCAATAAATTTGAGGTGCAAAAGGAGTTCATCAACCGGAACCTGGTGGA CACCCGGTACGCCACCAGAGAACTGACCAACTACCTGAAAGCCTACTTCAGCGCCAACA ACATGAACGTGAAGGTGAAAACCATCAACGGAAGCTTCACCGACTACCTGCGGAAGGTG

TGGAAGTTCAAGAAAGAGCGGAATCACGGCTATAAGCACCACGCTGAGGACGCTCTGAT CATCGCCAATGCCGATTTCCTGTTCAAGGAAAACAAGAAGTTAAAGGCCGTGAACTCTGT CCTGGAGAAGCCCGAGATCGAGACAAAGCAGCTGGACATCCAAGTGGACTCAGAGGAC

AATTACTCTGAGATGTTCATCATCCCCAAGCAGGTGCAGGACATCAAGGATTTTAGAAAT TTCAAGTACAGCCATAGAGTGGACAAGAAGCCCAATAGACAGCTGATCAACGATACACT GTACAGCACCAGAAAGAAGGACAACAGCACATATATCGTCCAGACCATCAAGGACATTT

ACGCAAAGGATAACACCACACTGAAGAAGCAGTTCGACAAAAGCCCTGAGAAGTTCCTG

ATGTACCAACACGACCCTCGGACCTTCGAGAAGTTAGAGGTTATCATGAAACAGTACGC

CAACGAGAAAAACCCTCTGGCCAAGTACCACGAGGAAACCGGAGAATACCTGACAAAA

TATAGCAAGAAGAACAACGGCCCCATCGTGAAAAGCCTGAAGTACATCGGCAACAAGCT GGGCAGCCACCTGGACGTGACCCACCAGTTCAAGAGCAGCACCAAGAAGCTGGTCAAGC TGAGCATCAAGCCTTATAGGTTCGACGTGTACCTGACAGATAAGGGATACAAGTTCATCA

CCATCAGCTACCTGGATGTTCTGAAAAAGGACAATTACTACTACATCCCTGAGCAGAAGT ACGACAAACTCAAGCTGGGCAAGGCCATCGATAAGAATGCCAAGTTCATCGCATCTTTTT ACAAGAACGACCTGATCAAACTGGACGGCGAGATCTACAAGATCATAGGAGTGAACAGC

GACACCAGGAATATGATCGAGCTCGATCTGCCTGACATCAGATACAAGGAATACTGCGA GCTGAACAACATCAAGGGAGAGCCTAGAATCAAGAAAACCATCGGCAAGAAGGTGAAC AGCATCGAGAAACTTACAACAGATGTGCTCGGCAACGTGTTCACCAACACCCAGTACAC

CAAGCCACAGCTGCTGTTTAAGCGGGGGAAC

[00235] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7028 (designated herein as Staphylococcus hyicus Cas9 or ShyCas9):

MNNYILGLDIGITSVGYGIVDSDTREIKDAGVRLFPEANVDNNEGRRSKRGARRLKR RRIHRLDRVKHLLAEYDLLDLTNIPKSTNPYQTRVKGLNEKLSKDELVIALLHIAKRRGIHNV NVMMDDNDSGNELSTKDQLKKNAKALSDKYVCELQLERFEQDYKVRGEKNRFKTEDFVRE ARKLLETQSKFFEIDQTFIMRYIELIETRREYFEGPGKGSPFGWEGNIKKWFEQMMGHCTYFP EELRSVKYSYSAELFNALNDLNNLVITRDEDAKLNYGEKFQIIENVFKQKKTPNLKQIAIEIGV HETEIKGYRVNKSGKPEFTQFKLYHDLKNIFKDPKYLNDIQLMDNIAEIITIYQDAESIIKELNQ LPELLSEREKEKISALSGYSGTHRLSLKCINLLLDDLWESSLNQMELFTKLNLKPKKIDLSQQH KIPSKLVDDFILSPVVKRAFIQSIQVVNAIIDKYGLPEDIIIELARENNSDDRRKFLNQLQKQNE ETRKQVEKVLREYGNDNAKRIVQKIKLHNMQEGKCLYSLKDIPLEDLLRNPHHYEVDHIIPRS VAFDNSMHNKVLVRADENSKKGNRTPYQYLNSSESSLSYNEFKQHILNLSKTKDRITKKKRE

YLLEERDINKFDVQKEFINRNLVDTRYATRELTSLLKAYFSANNLDVKVKTINGSFTNYLRKV

WKFDKDRNKGYKHHAEDALIIANADFLFKHNKKLRNINKVLDAPSKEVDKKRVTVQSEDEY NQIFEDTQKAQAIKKFEIRKFSHRVDKKPNRQLINDTLYSTRNIDGIEYVVESIKDIYSVNNDK VKTKFKKDPHRLLMYRNDPQTFEKFEKVFKQYESEKNPFAKYYEETGEKIRKFSKTGQGPYI NKIKYLRERLGRHCDVTNKYINSRNKIVQLKIYSYRFDIYQYGNNYKMITISYIDLEQKSNYY YISREKYEQKKKDKQIDDSYKFIGSFYKNDIINYNGEMYRVIGVNDSEKNKIQLDMIDISIKDY MELNNIKKTGVIYKTIGKSTTHIEKYTTDILGNLYKAAPPKKPQLIFK.

[00236] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7029 (designated herein as Staphylococcus microti Cas9 or Smi Cas9):

MEKDYILGLDIGIGSVGYGLIDYDTKSIIDAGVRLFPEANADNNLGRRAKRGARRLKRRRIHR

LERVKSLLSEYKIISGLAPTNNQPYNIRVKGLTEQLTKDELAVALLHIAKRRGIHNVDVAADK

EETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQ MQYYPEIDETFKEKYISLVETRREYYEGPGKGSPYGWDADVKKWYQLMMGHCTYFPVEFRS VKYAYTADLYNALNDLNNLTIARDDNPKLEYHEKYHIIENVFKQKRNPTLKQIAKEIGVNDI NISGYRVTKSGKPQFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLE YLMSEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGY QRIPTDMIDDAILSPVVKRSFKQAIGVVNAIIKKYGLPKDIIIELARESNSAEKSRYLRAIQKKN EKTRERIEAIIKEYGNENAKGLVQKIKLHDAQEGKCLYSLKDIPLEDLLRNPNNYDIDHIIPRS VSFDDSMHNKVLVRREQNAKKNNQTPYQYLTSGYADIKYSVFKQHVLNLAENKDRMTKKK REYLLEERNINKYDVQKEFINRNLVDTRYTTRELTTLLKTYFTINNLDVKVKTINGSFTDFLR KRWGFKKNRDEGYKHHAEDALIIANADYLFKEHKLLKEIKDVSDLAGDERNSNVKDEDQYE EVFGGYFKIEDIKKYKIKKFSHRVDKKPNRQLINDTIYSTRVKDDKRYLINTLKNLYDKSNGD

LKERMQKDPESLLMYHHDPQTFEKLKIVMSQYENEKNPLAKYFEETGQYLTKYAKHDNGPA IHKIKYYGNKLVEHLDITKNYHNPQNKVVQLSQKSFRFDVYQTDKGYKFISIAYLTLKNEKN YYAISQEKYDQLKSEKKISNNAVFIGSFYTSDIIEINNEKFRVIGVNSDKNNLIEVDRIDIRQKEF IELEEEKKNNRIKVTIGRKTTNIEKFHTDILGNMYKSKRPKAPQLVFKKG.

[00237] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7030 (designated herein as Staphylococcus pasteuri Cas9 or Spa Cas9):

MKEKYILGLDLGITSVGYGIINFETKKIIDAGVRLFPEANVDNNEGRRSKRGSRRLKRRRIHRL

ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNINVSSEDED

ASNELSTKEQINRNNKLLKDKYVCEVQLQRLKEGQIRGEKNRFKTTDILKEIDQLLKVQKDY

HNLDIDFINQYKEIVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYA YSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYR ITKSGTPQFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIYQDEQSIKEELNKLPEILNEQDK AEIAKLIGYNGTHRLSLKCIHLINEELWQTSRNQMEIFNYLNIKPNKVDLSEQNKIPKDMVND FILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIG QTGNQNAKRIVEKIRLHDQQEGKCLYSLESIALMDLLNNPQNYEVDHIIPRSVAFDNSIHNKV LVKQIENSKKGNRTPYQYLNSSDAKLSYNQFKQHILNLSKSKDRISKKKKDYLLEERDINKFE VQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRNHG YKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKEEDYNNVFETPKLVED IKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKKQFNKNPE KFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKV GNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQ ELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEINNIKG EPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[00238] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7031 (designated herein as Casl2il):

MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGIDRDIISG TANKDKISDDLLLAVNWFKVMPINSKPQGVSPSNLANLFQQYSGSEPDIQAQEYFASNFDTE KHQWKDMRVEYERLLAELQLSRSDMHHDLKLMYKEKCIGLSLSTAHYITSVMFGTGAKNN RQTKHQFYSKVIQLLEESTQINSVEQLASIILKAGDCDSYRKLRIRCSRKGATPSILKIVQDYEL GTNHDDEVNVPSLIANLKEKLGRFEYECEWKCMEKIKAFLASKVGPYYLGSYSAMLENALS PIKGMTTKNCKFVLKQIDAKNDIKYENEPFGKIVEGFFDSPYFESDTNVKWVLHPHHIGESNI KTLWEDLNAIHSKYEEDIASLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRY LYSRKDDIAVDKIIDGITFLSKKHKVEKQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLKHNL KCNRNQVDNYIWIEIKVLNTKTMRWEKHHYALSSTRFLEEVYYPATSENPPDALAARFRTKT NGYEGKPALSAEQIEQIRSAPVGLRKVKKRQMRLEAARQQNLLPRYTWGKDFNINICKRGN NFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIEAHANGDGVIDYNDLPVKPIESGF

VTVESQVRDKSYDQLSYNGVKLLYCKPHVESRRSFLEKYRNGTMKDNRGNNIQIDFMKDFE AIADDETSLYYFNMKYCKLLQSSIRNHSSQAKEYREEIFELLRDGKLSVLKLSSLSNLSFVMF KVAKSLIGTYFGHLLKKPKNSKSDVKAPPITDEDKQKADPEMFALRLALEEKRLNKVKSKKE VIANKIVAKALELRDKYGPVLIKGENISDTTKKGKKSSTNSFLMDWLARGVANKVKEMVM MHQGLEFVEVNPNFTSHQDPFVHKNPENTFRARYSRCTPSELTEKNRKEILSFLSDKPSKRPT NAYYNEGAMAFLATYGLKKNDVLGVSLEKFKQIMANILHQRSEDQLLFPSRGGMFYLATYK LDADATSVNWNGKQFWVCNADLVAAYNVGLVDIQKDFKKK.

[00239] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7032 (designated herein as Casl2i2):

MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQD IALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQ QYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKI LEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQ TDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFA KQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPAD PENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVL GNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAA GNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEIS ATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCF VRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVE AKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLS TLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIAN SLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGS LYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEF LSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVAT GAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS.

Modified guide RNAs

[00240] In some embodiments, the guide RNA is chemically modified. A guide RNA comprising one or more modified nucleosides or nucleotides is called a “modified” guide RNA or “chemically modified” guide RNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified guide RNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.” Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3' or 5' cap modifications may comprise a sugar and/or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification).

[00241] Chemical modifications such as those listed above can be combined to provide modified guide RNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase, or a modified sugar and a modified phosphodiester. In some embodiments, every base of a guide RNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of a guide RNA molecule are replaced with phosphorothioate groups. In some embodiments, modified guide RNAs comprise at least one modified residue at or near the 5' end of the RNA. In some embodiments, modified guide RNAs comprise at least one modified residue at or near the 3' end of the RNA.

[00242] In some embodiments, the guide RNA comprises one, two, three or more modified residues. In some embodiments, at least 5% e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in a modified guide RNA are modified nucleosides or nucleotides.

[00243] Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the guide RNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified guide RNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[00244] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[00245] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

[00246] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

[00247] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[00248] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g. replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion.

[00249] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_nCH₂CH₂OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2' hydroxyl group modification can be 2'-0-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a Ci-6 alkylene or Ci-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH₂)_n-amino, (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can include "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, e.g., a PEG derivative).

[00250] “Deoxy” 2' modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_nCH2CH₂- amino (wherein amino can be, e.g., as described herein), -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein. [00251] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides.

[00252] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

[00253] In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA. In embodiments comprising sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, and/or internal nucleosides may be modified, and/or the entire sgRNA may be chemically modified. Certain embodiments comprise a 5' end modification. Certain embodiments comprise a 3' end modification.

[00254] Modifications of 2’-O-methyl are encompassed.

[00255] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2’ -fluoro (2’-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability. Modifications of 2’ -fluoro (2’-F) are encompassed.

[00256] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.

[00257] Abasic nucleotides refer to those which lack nitrogenous bases.

[00258] Inverted bases refer to those with linkages that are inverted from the normal 5’ to 3’ linkage (i.e., either a 5’ to 5’ linkage or a 3’ to 3’ linkage).

[00259] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5’ nucleotide via a 5’ to 5’ linkage, or an abasic nucleotide may be attached to the terminal 3’ nucleotide via a 3’ to 3’ linkage. An inverted abasic nucleotide at either the terminal 5’ or 3’ nucleotide may also be called an inverted abasic end cap.

[00260] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' terminus, and one or more of the last three, four, or five nucleotides at the 3' terminus are modified. In some embodiments, the modification is a 2’-0-Me, 2’-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability and/or performance.

[00261] In some embodiments, the first four nucleotides at the 5' terminus, and the last four nucleotides at the 3' terminus are linked with phosphorothioate (PS) bonds.

[00262] In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-O-methyl (2'-0-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-fluoro (2'-F) modified nucleotide.

Ribonucleoprotein complex

[00263] In some embodiments, a composition is encompassed comprising: a) one or more guide RNAs comprising one or more guide sequences or one or more guide pairs from Tables 1A-1F and b) SluCas9. In some embodiments, the guide RNA together with a Cas9 is called a ribonucleoprotein complex (RNP).

[00264] In some embodiments, the disclosure provides for an RNP complex, wherein the guide RNA (e.g., any of the guide RNAs disclosed herein) binds to or is capable of binding to a target sequence in the dystrophin gene.

[00265] In some embodiments, chimeric Cas9 (SaCas9 or SluCas9) nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas9 nuclease domain may be replaced with a domain from a different nuclease such as Fokl. In some embodiments, a Cas9 nuclease may be a modified nuclease. [00266] In some embodiments, the Cas9 is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.

[00267] In some embodiments, a conserved amino acid within a Cas9 protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas9 nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3): 759-771. In some embodiments, the Cas9 nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpfl (FnCpfl) sequence (UniProtKB - A0Q7Q2 (CPF1_FRATN)). Further exemplary amino acid substitutions include D10A and N580A (based on the S. aureus Cas9 protein). See, e.g., Friedland et al., 2015, Genome Biol., 16:257.

[00268] In some embodiments, the Cas9 lacks cleavase activity. In some embodiments, the Cas9 comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the Cas9 lacking cleavase activity or the dCas DNA- binding polypeptide is a version of a Cas nuclease (e.g., a Cas9 nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 2014/0186958 Al; US 2015/0166980 Al.

[00269] In some embodiments, the Cas9 comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[00270] In some embodiments, the heterologous functional domain may facilitate transport of the Cas9 into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the Cas9 may be fused with 1-10 NLS(s). In some embodiments, the Cas9 may be fused with 1-5 NLS(s). In some embodiments, the Cas9 may be fused with 1-3 NLS(s). In some embodiments, the Cas9 may be fused with one NLS. Where one NLS is used, the NLS may be attached at the N-terminus or the C-terminus of the Cas9 sequence, and may be directly fused/attached. In some embodiments, where more than one NLS is used, one or more NLS may be attached at the N-terminus and/or one or more NLS may be attached at the C-terminus. In some embodiments, one or more NLSs are directly attached to the Cas9. In some embodiments, one or more NLSs are attached to the Cas9 by means of a linker. In some embodiments, the linker is between 3-25 amino acids in length. In some embodiments, the linker is between 3-6 amino acids in length. In some embodiments, the linker comprises glycine and serine. In some embodiments, the linker comprises the sequence of GSVD (SEQ ID NO: 550) or GSGS (SEQ ID NO: 551). It may also be inserted within the Cas9 sequence. In other embodiments, the Cas9 may be fused with more than one NLS. In some embodiments, the Cas9 may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the Cas9 may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the Cas9 protein is fused with one or more SV40 NLSs. In some embodiments, the SV40 NLS comprises the amino acid sequence of SEQ ID NO: 713 (PKKKRKV). In some embodiments, the Cas9 protein (e.g., the SaCas9 or SluCas9 protein) is fused to one or more nucleoplasmin NLSs. In some embodiments, the Cas protein is fused to one or more c-myc NLSs. In some embodiments, the Cas protein is fused to one or more E1A NLSs. In some embodiments, the Cas protein is fused to one or more BP (bipartite) NLSs. In some embodiments, the nucleoplasmin NLS comprises the amino acid sequence of SEQ ID NO: 714 (KRPAATKKAGQAKKKK). In some embodiments, the Cas9 protein is fused with a c-Myc NLS. In some embodiments, the c-Myc NLS comprises the amino acid sequence of SEQ ID NO: 942 (PAAKKKKLD) and/or is encoded by the nucleic acid sequence of SEQ ID NO: 722 (CCGGCAGCTAAGAAAAAGAAACTGGAT). In some embodiments, the Cas9 is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the Cas9 may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the Cas9 may be fused with 3 NLSs. In some embodiments, the Cas9 may be fused with 3 NLSs, two linked at the N-terminus and one linked at the C-terminus. In some embodiments, the Cas9 may be fused with 3 NLSs, one linked at the N-terminus and two linked at the C-terminus. In some embodiments, the Cas9 may be fused with no NLS. In some embodiments, the Cas9 may be fused with one NLS. In some embodiments, the Cas9 may be fused with an NLS on the C-terminus and does not comprise an NLS fused on the N-terminus. In some embodiments, the Cas9 may be fused with an NLS on the N-terminus and does not comprise an NLS fused on the C-terminus. In some embodiments, the Cas9 protein is fused to an SV40 NLS and to a nucleoplasmin NLS. In some embodiments, the Cas9 protein is fused to an SV40 NLS and to a c-Myc NLS. In some embodiments, the SV40 NLS is fused to the C-terminus of the Cas9, while the nucleoplasmin NLS is fused to the N- terminus of the Cas9 protein. In some embodiments, the SV40 NLS is fused to the C-terminus of the Cas9, while the c-Myc NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the SV40 NLS is fused to the N-terminus of the Cas9, while the nucleoplasmin NLS is fused to the C- terminus of the Cas9 protein. In some embodiments, the SV40 NLS is fused to the N-terminus of the Cas9, while the c-Myc NLS is fused to the C-terminus of the Cas9 protein. In some embodiments, the SV40 NLS is fused to the Cas9 protein by means of a linker. In some embodiments, the SV40 NLS and linker is encoded by the nucleic acid sequence of SEQ ID NO: 723 ( ATGATGGCCCC A A AGA AGA AGCGGA AGGTCGGT ATCC ACGGAGTCCC AGC AGCC) . In some embodiments, the nucleoplasmin NLS is fused to the Cas9 protein by means of a linker. In some embodiments, the c-Myc NLS is fused to the Cas9 protein by means of a linker. In some embodiments, an additional domain may be: a) fused to the N- or C-terminus of the Cas protein (e.g., a Cas9 protein), b) fused to the N-terminus of an NLS fused to the N-terminus of a Cas protein, or c) fused to the C-terminus of an NLS fused to the C-terminus of a Cas protein. In some embodiments, an NLS is fused to the N- and/or C-terminus of the Cas protein by means of a linker. In some embodiments, an NLS is fused to the N-terminus of an N-terminally-fused NLS on a Cas protein by means of a linker, and/or an NLS is fused to the C-terminus of a C-terminally fused NLS on a Cas protein by means of a linker. In some embodiments, the linker is GSVD (SEQ ID NO: 550) or GSGS (SEQ ID NO: 551). In some embodiments, the Cas protein comprises a c-Myc NLS fused to the N- terminus of the Cas protein (or to an N-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises an SV40 NLS fused to the C- terminus of the Cas protein (or to a C-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises a nucleoplasmin NLS fused to the C- terminus of the Cas protein (or to a C-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N- terminus of the Cas protein, optionally by means of a linker, b) an SV40 NLS fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) a nucleoplasmin NLS fused to the C- terminus of the SV40 NLS, optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N-terminus of the Cas protein, optionally by means of a linker, b) a nucleoplasmin NLS fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) an SV40 NLS fused to the C-terminus of the nucleoplasmin NLS, optionally by means of a linker. In some embodiments, a c-myc NLS is fused to the N-terminus of the Cas9 and an SV40 NLS and/or nucleoplasmin NLS is fused to the C-terminus of the Cas9. In some embodiments, a c- myc NLS is fused to the N-terminus of the Cas9 (e.g., by means of a linker such as GSVD), an SV40 NLS is fused to the C-terminus of the Cas9 (e.g., by means of a linker such as GSGS), and a nucleoplasmin NLS is fused to the C-terminus of the SV-40 NLS (e.g., by means of a linker such as GSGS).

[00271] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the Cas9. In some embodiments, the half-life of the Cas9 may be increased. In some embodiments, the half-life of the Cas9 may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the Cas9. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the Cas9. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the Cas9 may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rubl in 5. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBF (MUB), ubiquitin fold- modifier-1 (UFM1), and ubiquitin-like protein-5 (UBF5).

[00272] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, betaglucuronidase, luciferase, or fluorescent proteins.

[00273] In additional embodiments, the heterologous functional domain may target the Cas9 to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the Cas9 to muscle.

[00274] In further embodiments, the heterologous functional domain may be an effector domain. When the Cas9 is directed to its target sequence, e.g., when a Cas9 is directed to a target sequence by a guide RNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain or a nuclease domain (e.g., a non-Cas nuclease domain). In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., US Pat. No. 9,023,649.

[00275] In some embodiments, any of the compositions disclosed herein comprising any of the guides and/or endonucleases disclosed herein is sterile and/or substantially pyrogen-free. In particular embodiments, any of the compositions disclosed herein comprise a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein “pharmaceutically acceptable carrier” includes any and all solvents (e.g., water), dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, including pharmaceutically acceptable cell culture media. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In some embodiments, the composition comprises a preservative to prevent the growth of microorganisms.

Determination of efficacy of guide RNAs

[00276] In some embodiments, the efficacy of a guide RNA is determined when delivered or expressed together with other components forming an RNP. In some embodiments, the guide RNA is expressed together with a SaCas9 or SluCas9 or sRGN. In some embodiments, the guide RNA is delivered to or expressed in a cell line that already stably expresses an SaCas9 or SluCas9. In some embodiments the guide RNA is delivered to a cell as part of an RNP. In some embodiments, the guide RNA is delivered to a cell along with a nucleic acid (e.g., mRNA) encoding SaCas9 or SluCas9. [00277] In some embodiments, the efficacy of particular guide RNAs is determined based on in vitro models. In some embodiments, the in vitro model is a cell line.

[00278] In some embodiments, the efficacy of particular guide RNAs is determined across multiple in vitro cell models for a guide RNA selection process. In some embodiments, a cell line comparison of data with selected guide RNAs is performed. In some embodiments, cross screening in multiple cell models is performed.

[00279] In some embodiments, the efficacy of particular guide RNAs is determined based on in vivo models. In some embodiments, the in vivo model is a rodent model. In some embodiments, the rodent model is a mouse which expresses a mutated dystrophin gene. In some embodiments, the in vivo model is a non-human primate, for example cynomolgus monkey.

III. Methods of Gene Editing and Treating DMD

[00280] This disclosure provides methods for gene editing and treating Duchenne Muscular Dystrophy (DMD). In some embodiments, any of the compositions described herein may be administered to a subject in need thereof for use in excising all or a portion (e.g., less than about 400 nucleotides) of exon 53 of the dystrophin (DMD) gene and to treat DMD. In some embodiments, pairs of guide RNAs described herein, in any of the vector configurations described herein, may be administered to a subject in need thereof to excise all or a portion of exon 53 of the DMD gene, and treat DMD. In some embodiments, any of the compositions described herein may be administered to a subject in need thereof for use in treating DMD. In some embodiments, a nucleic acid molecule comprising a first nucleic acid encoding one or more guide RNAs of Tables 1A-1C and a second nucleic acid encoding SluCas9 or another Cas protein (e.g., a sRGN) is administered to a subject to treat DMD. In some embodiments, a single nucleic acid molecule (which may be a vector, such as an AAV vector) comprising a first nucleic acid encoding one or more guide RNAs of Tables 1A-1C and a second nucleic acid encoding either SluCas9 is administered to a subject to treat DMD. In some embodiments, more than one nucleic acid molecules (which may be a vector, such as an AAV vectors) are administered to a subject to treat DMD, wherein a first nucleic acid encoding one or more guide RNAs of Tables 1A-1C and a second nucleic acid encoding either SluCas9 or another Cas protein (e.g., a sRGN) are together on one nucleic acid molecule or separated on different nucleic acid molecules.

[00281] In some embodiments, any of the compositions described herein is administered to a subject in need thereof to treat DMD.

[00282] In some embodiments, any of the compositions described herein is administered to a subject in need thereof to induce a double or single strand break in exon 53 of the dystrophin gene.

[00283] In some embodiments, any of the compositions described herein is administered to a subject in need thereof to delete all or a portion (e.g., excise a portion) of exon 53 of the dystrophin gene, such as all of exon 53 of the dystrophin gene, a portion of exon 53 flanking a splice acceptor site, or a portion of exon 53 flanking a splice donor site.

[00284] In some embodiments, a method of treating DMD is provided, the method comprising delivering to a cell any one of the compositions described herein, wherein the cell comprises a mutation in the dystrophin gene that is known to be associated with DMD.

[00285] In some embodiments, a method for treating DMD is provided comprising administering a composition comprising one or more guide RNAs wherein each guide RNA comprises a guide sequence of Tables 1A-1C, or comprises a guide sequence comprising at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to guide sequence selected from Tables 1A-1C. In each instance, the composition may comprise a nucleic acid encoding the guide RNA(s). In some embodiments, a method for treating DMD is provided comprising administering a composition comprising one or more nucleic acid molecules encoding one or more guide RNAs, wherein each guide RNA comprises a guide sequence of Tables 1A-1C, or at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 90% identical to guide sequence selected from Tables 1A-1C.

[00286] In some embodiments, a method for treating DMD is provided comprising administering a composition comprising a guide RNA wherein the guide RNA comprises a guide sequence of Tables 1A-1C, or at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to guide sequence of Tables 1A-1C. In each instance, the composition may comprise a nucleic acid encoding the guide RNA.

[00287] In some embodiments, a method for treating DMD is provided comprising administering a composition comprising a guide RNA comprising no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 contiguous nucleotides of a guide sequence of Tables 1A-1C. In each instance, the composition may comprise a nucleic acid encoding the guide RNA.

[00288] In some embodiments, a method for treating DMD is provided comprising administering a composition comprising two or more guide RNAs wherein each guide RNA comprises a guide sequence of Tables 1A-1C, or at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of Tables 1A-1C, or is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to guide sequence selected from Tables 1A-1C. In each instance, the composition may comprise a nucleic acid encoding the guide RNA.

[00289] In some embodiments, a method for treating DMD is provided comprising administering a composition comprising two or more guide RNAs wherein at least one of the two or more guide RNAs, optionally each guide RNA, comprises no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 contiguous nucleotides of a guide sequence of Tables 1A-1C, is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 contiguous nucleotides of a guide sequence selected from Tables 1A-1C. In each instance, the composition may comprise a nucleic acid encoding the guide RNA.

[00290] In some embodiments, a method for treating DMD is provided comprising administering a composition comprising a SluCas9 or sRGN protein or a nucleic acid encoding a SluCas9 or sRGN protein and a pair of guide RNAs, wherein the pair of guide RNAs comprise or consist of any one of the pairs of guide sequences of any one of Tables 1D-1F.

[00291] In some embodiments, a method for treating DMD is provided comprising administering a SluCas9 or sRGN protein or a nucleic acid encoding a SluCas9 or sRGN protein and a composition comprising one or more nucleic acid molecules encoding a pair of guide RNAs, wherein the pair of guide RNAs comprise or consist of one or more nucleic acid molecules at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the pairs of guide sequences of any one of Tables 1D-1F. [00292] In some embodiments, methods are provided for treating DMD, the method comprising delivering to a cell at least one nucleic acid molecule comprising a pair of guide RNAs comprising a first and second guide RNA, wherein the first guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 1-64; and the second guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 88-143.

[00293] In some embodiments, methods are provided for treating DMD, the method comprising delivering to a cell at least one nucleic acid molecule comprising a pair of guide RNAs comprising a first and second spacer sequence, wherein the pair of first and spacer sequences are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequences of any one of the following pairs: a. a pair of guide sequences selected from Table ID; or b. SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28; 113 and 35;

113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48; 113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and 16; 113 and 17; 113 and 57; 113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and 23; 113 and 24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48;

115 and 13; 115 and 53; 115 and 54; 115 and 55; 115 and 56; 115 and 14; 115 and 15; 115 and

16; 115 and 17; 115 and 57; 115 and 18; 115 and 58; 115 and 19; 115 and 20; 115 and 59; 115 and 23; 115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and 26;

116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42; and a nucleic acid encoding a SluCas9, wherein the nucleic acid encoding the SluCas9 may be on the same or different nucleic acid molecule as the pair of guide RNAs.

[00294] In some embodiments, methods are provided for treating DMD, the method comprising delivering to a cell at least one nucleic acid molecule comprising a pair of guide RNAs comprising a first and second guide RNA, wherein the first guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 1-64; and the second guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 65-87. In some embodiments, methods are provided for treating DMD, the method comprising delivering to a cell at least one nucleic acid molecule comprising a pair of guide RNAs comprising a first and second guide RNA, wherein the first guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 65-87; and the second guide RNA comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical any one of SEQ ID NOs: 88-143.

[00295] In some embodiments, methods are provided for treating DMD, the method comprising delivering to a cell at least one nucleic acid molecule comprising a pair of guide RNAs comprising a first and second spacer sequence, wherein the pair of first and spacer sequences are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequences of any one of the following pairs: a. a pair of guide sequences selected from Table IE or Table 1F. b. SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and 80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85;

19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70; 57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80;

20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76; 58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; or c. SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82; 121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and

83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and 84; 119 and 84; 120 and 84; 121 and

84; 90 and 84; 115 and 85; 116 and 85; 117 and 85; 118 and 85; 119 and 85; 120 and 85; 121 and

85; 90 and 85; 115 and 68; 116 and 68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and

68; 90 and 68; 115 and 69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and

69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and

70; 90 and 70; 115 and 71; 116 and 71; 117 and 71; 118 and 71; 119 and 71; 120 and 71; 121 and

71; 90 and 71; 115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and

73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and

74; 90 and 74; 115 and 75; 116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and

75; 90 and 75; 115 and 76; 116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and

76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and

80; 90 and 80; 115 and 81; 116 and 81; 117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and

81 ; or 90 and 81 ; and a nucleic acid encoding a SluCas9, wherein the nucleic acid encoding the SluCas9 may be on the same or different nucleic acid molecule as the pair of guide RNAs.

[00296] Also provided are methods of excising a fragment of a DMD gene, comprising delivering to a cell a composition comprising (a) a pair of guide RNA molecules or a nucleic acid encoding a pair of RNA molecules, wherein the first guide RNA targets a region upstream of a splice acceptor site or a splice donor site in a DMD gene and wherein the second guide RNA targets a region downstream of a splice acceptor site or a splice donor site in the DMD gene, and (b) an endonuclease or a nucleic acid encoding an endonuclease. In some embodiments, the endonuclease is a Staphylococcus lugdunensis Cas9 (SluCas9). In one particular embodiment, the first guide RNA is in an intron of the DMD gene and the second guide RNA is in an exon of the DMD gene. In another particular embodiment, the first guide RNA is in an exon of the DMD gene and the second guide RNA is in an intron of the DMD gene. In another particular embodiment, the first guide RNA is in an exon of the DMD gene and the second guide RNA is in an exon of the DMD gene. In another particular embodiment, the first guide RNA is in an intron of the DMD gene and the second guide RNA is in an intron of the DMD gene. In some embodiments, the pair of guide RNAs and the endonuclease excise a fragment of the DMD gene comprising the splice acceptor site or the splice donor site, wherein the fragment is between 3 and 400 nucleotides in length. In some embodiments, the excised fragment of the DMD gene comprises a splice acceptor site. In other embodiments, the excised fragment of the DMD gene comprises a splice donor site. In some embodiments, the excised fragment is between 3-200, 3-150, 3-100, 3-50, 30-250, 30-200, 30-150, 30-100, 30-50, 75-250, 75- 200, 75-150, 75-100, 100-250, 100-200, 100-150, 150-250, 150-200, 200-250, 250-400, 300-400, or 350-400 nucleotides in length. [00297] In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[00298] For treatment of a subject (e.g., a human), any of the compositions disclosed herein may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The compositions may be readily administered in a variety of dosage forms, such as injectable solutions. For parenteral administration in an aqueous solution, for example, the solution will generally be suitably buffered and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, and/or intraperitoneal administration.

Combination Therapy

[00299] In some embodiments, the invention comprises combination therapies comprising any of the methods or uses described herein together with an additional therapy suitable for ameliorating DMD.

Delivery of Guide RNA Compositions

[00300] The methods and uses disclosed herein may use any suitable approach for delivering the guide RNAs and compositions described herein. Exemplary delivery approaches include vectors, such as viral vectors; lipid nanoparticles (LNPs); liposomes; transfection; and electroporation. In some embodiments, vectors, such as viral vectors, LNPs, or liposomes associated with the single-vector guide RNAs/Cas9’s disclosed herein are for use in preparing a medicament for treating DMD.

[00301] Lipid nanoparticles (LNPs) are a known means for delivery of nucleotide and protein cargo, and may be used for delivery of the guide RNAs, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.

[00302] Electroporation is a well-known means for delivery of cargo, and any electroporation methodology may be used for delivering the single vectors disclosed herein.

[00303] In some embodiments, the invention comprises a method for delivering any one of the single vectors disclosed herein to an ex vivo cell, wherein the guide RNA is encoded by a vector, associated with an LNP or a liposome, or in aqueous solution. In some embodiments, the guide RNA/LNP, guide RNA/liposome, or guide RNA is also associated with a Cas9 (such as a SluCas9) or sequence encoding Cas9 (e.g., in the same vector (such as the same viral vector, LNP, or liposome), or solution).

[00304] In some embodiments, the disclosure provides for methods of using any of the guides, endonucleases, cells, or compositions disclosed herein in research methods. For example, any of the guides or endonucleases disclosed herein may be used alone or in combination in experiments under various parameters (e.g., temperatures, pH, types of cells) or combined with other reagents to evaluate the activity of the guides and/or endonucleases.

EXAMPLES

[00305] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.

Example 1: Exemplary DMD sgRNAs

[00306] Guide RNAs comprising the guide sequences shown in Tables 1A-1C (SEQ ID NOs: 1- 143) are prepared according to standard methods in a single guide (sgRNA) format. A single AAV vector, or two AAV vectors, are prepared that express one or more of the guide RNAs and a SluCas9. The AAV vector is administered to cells in vitro to assess the ability of the AAV to express the one or more guide RNAs and SluCas9, edit the targeted region of exon 53, and thereby treat DMD.

Example 2: Exemplary DMD sgRNA pairs

[00307] Guide RNAs comprising the guide sequences shown in Tables 1A-1C (SEQ ID NOs: 1- 143) are prepared according to standard methods in a single guide (sgRNA) format. A single AAV vector, or two AAV vectors, are prepared that express one or more guide RNA pairs selected from Tables 1D-1F (see, Figure 4), and a SluCas9. The AAV vector is administered to cells in vitro to assess the ability of the AAV to express the one or more guide RNA pairs and SluCas9, excise the targeted region of exon 53, and thereby treat DMD.

Example 3: Evaluation of sgRNA Pairs

[00308] Immortalized human skeletal muscle myoblast cells (iHsMM) comprising either the full DMD gene sequence (WT13) or with the DMD exon 52 deleted (AE52) were used for in vitro evaluation of sgRNA pairs. Cells were transfected with SluCas9 mRNA (or in some cases sRGN3.3 mRNA) along with synthetic sgRNAs using the Lipofectamine MessengerMAX reagent.

[00309] For qPCR, cells were seeded into 96-well plates after transfection and were harvested after 72 hours. Genomic DNA (gDNA) was extracted from each sample using the MagMAX DNA Ultra 2.0 kit, followed by qPCR analysis. qPCR was performed on each gDNA sample separately with an “Editing” primer pair that amplified a portion of exon 53 and a “Control” primer pair that amplified a portion of intron 52. The differences in the cycle number of the “Editing” pair and the “Control” pair was used to calculate the percentage of exon 53 deletion for each gDNA sample.

[00310] For dystrophin/Jess protein analyses, cells were seeded into 12-well plates coated with Matrigel after transfection. After 48 hours, cells were then maintained in Myocult media for 12 days to induce differentiation into myotubes and to induce expression of dystrophin. Cells were then harvested and lysed using MSD buffer and the protein concentration was measured using Pierce BCA (ThermoFisher, 23227) assay according to the manufacturer's protocol. Next, samples were prepared for Jess Separation module (“Jess”), according to the manufacturer’s protocol. Primary antibody (abl54168, Abeam) was used for dystrophin protein detection at 1:250 dilution, and secondary antibody (ProteinSimple, 042-206) and Total Protein Detection Module DM-TP01 were used for normalizing the samples during analysis. Samples were run using 24-well cartridges and 66-440 kDa kits for Jess analysis. The data were acquired using the capillary gel electrophoresis system ProteinSimple “Jess” and analyzed using Compass for Simple Western software.

[00311] Figure 1 shows that tested guide pairs targeting intron 52 and intron 53 of the dystrophin gene result in different excision fragment sizes, e.g., from about 500-600 base pairs in length to about 200-300 bp in length. Figure 2 shows the editing frequencies of selected SluCas9 guide pairs targeting intron 52 and intron 53 of the dystrophin gene, for excision of exon 53 in the immortalized HsMM AE52 cells. Editing frequencies for the experimental pairs ranged from approximately 15% to more than 40%. Figures 3A and 3B show dystrophin restoration in immortalized HsMM AE52 cells treated with selected SluCas9 sgRNA pairs. Figures 3A and 3B detection of dystrophin restoration in immortalized HsMM AE52 cells treated with selected SluCas9 sgRNA pairs that result in either precise reframing deletion or deletion of the entire exon 53 of the dystrophin gene, using the Jess protein assay (Figure 3A) or qPCR (Figure 3B). All guide pairs tested restored dystrophin levels to 30-50% of control levels on day 12. Each bar in Figure 3B represents a sgRNA pair and the bar height depicts normalized dystrophin protein signal of the sample as a percentage of normalized dystrophin expression in untreated WT13 cells. Figures 5 and 6 show dystrophin restoration in immortalized HsMM AE52 cells treated with selected SluCas9 or sRGN sgRNA pairs that result in either precise reframing deletion, deletion of the entire exon 53 of the dystrophin gene, or skipping of exon 53. Each bar represents a sgRNA pair or triplet and the bar height depicts dystrophin protein signal normalized to the total protein signal, as determined by Jess protein assay. 19

Claims

What is claimed is:

1. A composition comprising one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise i) a guide sequence of any one of the guide sequences shown in Tables 1A or 1C; ii) at least 16, 17, 18, 19, or 20 contiguous nucleotides of any one of the guide sequences shown in Tables 1A or 1C; iii) a guide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the guide sequences shown in Tables 1A or 1C; or iv) a pair of guide RNAs comprising any of the guide sequence pairs shown in Table ID, optionally further comprising a Staphylococcus lugdunensis Cas9 (SluCas9) or a nucleic acid encoding a SluCas9.

2. A composition comprising a first guide RNA and a second guide RNA, wherein a. the first guide RNA is selected from any one of SEQ ID NOs: 1-64; and b. the second guide RNA is selected from any one of SEQ ID NOs: 88-143.

3. A composition comprising at least one pair of guide RNAs or a nucleic acid encoding at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprise or consist of a first and a second guide sequence selected from any of the following pairs of first and second guide sequences: a. SEQ ID NOs: 88 and 1; 88 and 2; 88 and 3; 88 and 4; 88 and 5; 88 and 6; 88 and 7; 88 and 8; 88 and 9; 88 and 10; 88 and 11; 88 and 12; 88 and 13; 88 and 14; 88 and 15; 88 and 16; 88 and 17; 88 and 18; 88 and 19; 88 and 20; 88 and 21; 88 and 22; 88 and 23; 88 and 24; 88 and 25; 88 and 26; 88 and 27; 88 and 28; 88 and 29; 88 and 30; 88 and 31; 88 and 32; 88 and 33; 88 and 34; 88 and 35; 88 and 36; 88 and 37; 88 and 38; 88 and 39; 88 and 40; 88 and 41; 88 and 42; 88 and 43; 88 and 44; 88 and 45; 88 and 46; 88 and 47; 88 and 48; 88 and 49; 88 and 50; 88 and 51; 88 and 52; 88 and 53;

88 and 54; 88 and 55; 88 and 56; 88 and 57; 88 and 58; 88 and 59; 88 and 60; 88 and 61; 88 and 62; 88 and 63; 88 and 64; 89 and 1; 89 and 2; 89 and 3; 89 and 4; 89 and 5; 89 and 6; 89 and 7; 89 and 8; 89 and 9; 89 and 10; 89 and 11; 89 and 12; 89 and 13; 89 and 14; 89 and 15; 89 and 16; 89 and 17; 89 and 18; 89 and 19; 89 and 20; 89 and 21; 89 and 22; 89 and 23; 89 and 24; 89 and 25; 89 and 26; 89 and 27; 89 and 28;

89 and 29; 89 and 30; 89 and 31; 89 and 32; 89 and 33; 89 and 34; 89 and 35; 89 and 36; 89 and 37; 89 and 38; 89 and 39; 89 and 40; 89 and 41; 89 and 42; 89 and 43; 89 and 44; 89 and 45; 89 and 46; 89 and 47; 89 and 48; 89 and 49; 89 and 50; 89 and 51; 89 and 52; 89 and 53; 89 and 54; 89 and 55; 89 and 56; 89 and 57; 89 and 58; 89 and 59; 89 and 60; 89 and 61; 89 and 62; 89 and 63; 89 and 64; 90 and 1; 90 and 2; 90 and 3; 90 and 4; 90 and 5; 90 and 6; 90 and 7; 90 and 8; 90 and 9; 90 and 10; 90 and 11; 90 and 12; 90 and 13; 90 and 14; 90 and 15; 90 and 16; 90 and 17; 90 and 18; 90 and 19; 90 and 20; 90 and 21; 90 and 22; 90 and 23; 90 and 24; 90 and 25; 90 and 26; 90 and 27; 90 and 28; 90 and 29; 90 and 30; 90 and 31; 90 and 32; 90 and 33; 90 and 34; 90 and 35; 90 and 36; 90 and 37; 90 and 38; 90 and 39; 90 and 40; 90 and 41; 90 and 42; 90 and 43; 90 and 44; 90 and 45; 90 and 46; 90 and 47; 90 and 48; 90 and 49;

90 and 50; 90 and 51; 90 and 52; 90 and 53; 90 and 54; 90 and 55; 90 and 56; 90 and 57; 90 and 58; 90 and 59; 90 and 60; 90 and 61; 90 and 62; 90 and 63; 90 and 64; 91 and 1; 91 and 2; 91 and 3; 91 and 4; 91 and 5; 91 and 6; 91 and 7; 91 and 8; 91 and 9;

91 and 10; 91 and 11; 91 and 12; 91 and 13; 91 and 14; 91 and 15; 91 and 16; 91 and 17; 91 and 18; 91 and 19; 91 and 20; 91 and 21; 91 and 22; 91 and 23; 91 and 24; 91 and 25; 91 and 26; 91 and 27; 91 and 28; 91 and 29; 91 and 30; 91 and 31; 91 and 32; 91 and 33; 91 and 34; 91 and 35; 91 and 36; 91 and 37; 91 and 38; 91 and 39; 91 and 40; 91 and 41; 91 and 42; 91 and 43; 91 and 44; 91 and 45; 91 and 46; 91 and 47; 91 and 48; 91 and 49; 91 and 50; 91 and 51; 91 and 52; 91 and 53; 91 and 54; 91 and 55;

91 and 56; 91 and 57; 91 and 58; 91 and 59; 91 and 60; 91 and 61; 91 and 62; 91 and 63; 91 and 64; 92 and 1; 92 and 2; 92 and 3; 92 and 4; 92 and 5; 92 and 6; 92 and 7;

92 and 8; 92 and 9; 92 and 10; 92 and 11; 92 and 12; 92 and 13; 92 and 14; 92 and 15; 92 and 16; 92 and 17; 92 and 18; 92 and 19; 92 and 20; 92 and 21; 92 and 22; 92 and 23; 92 and 24; 92 and 25; 92 and 26; 92 and 27; 92 and 28; 92 and 29; 92 and 30; 92 and 31; 92 and 32; 92 and 33; 92 and 34; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 92 and 43; 92 and 44; 92 and 45; 92 and 46; 92 and 47; 92 and 48; 92 and 49; 92 and 50; 92 and 51; 92 and 52; 92 and 53;

92 and 54; 92 and 55; 92 and 56; 92 and 57; 92 and 58; 92 and 59; 92 and 60; 92 and 61; 92 and 62; 92 and 63; 92 and 64; 93 and 1; 93 and 2; 93 and 3; 93 and 4; 93 and 5; 93 and 6; 93 and 7; 93 and 8; 93 and 9; 93 and 10; 93 and 11; 93 and 12; 93 and 13; 93 and 14; 93 and 15; 93 and 16; 93 and 17; 93 and 18; 93 and 19; 93 and 20; 93 and 21; 93 and 22; 93 and 23; 93 and 24; 93 and 25; 93 and 26; 93 and 27; 93 and 28;

93 and 29; 93 and 30; 93 and 31; 93 and 32; 93 and 33; 93 and 34; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; 93 and 42; 93 and 43; 93 and 44; 93 and 45; 93 and 46; 93 and 47; 93 and 48; 93 and 49; 93 and 50; 93 and 51;

93 and 52; 93 and 53; 93 and 54; 93 and 55; 93 and 56; 93 and 57; 93 and 58; 93 and 59; 93 and 60; 93 and 61; 93 and 62; 93 and 63; 93 and 64; 94 and 1; 94 and 2; 94 and 3; 94 and 4; 94 and 5; 94 and 6; 94 and 7; 94 and 8; 94 and 9; 94 and 10; 94 and 11; 94 and 12; 94 and 13; 94 and 14; 94 and 15; 94 and 16; 94 and 17; 94 and 18; 94 and 19; 94 and 20; 94 and 21; 94 and 22; 94 and 23; 94 and 24; 94 and 25; 94 and 26;

94 and 27; 94 and 28; 94 and 29; 94 and 30; 94 and 31; 94 and 32; 94 and 33; 94 and 34; 94 and 35; 94 and 36; 94 and 37; 94 and 38; 94 and 39; 94 and 40; 94 and 41; 94 and 42; 94 and 43; 94 and 44; 94 and 45; 94 and 46; 94 and 47; 94 and 48; 94 and 49;

94 and 50; 94 and 51; 94 and 52; 94 and 53; 94 and 54; 94 and 55; 94 and 56; 94 and 57; 94 and 58; 94 and 59; 94 and 60; 94 and 61; 94 and 62; 94 and 63; 94 and 64; 95 and 1; 95 and 2; 95 and 3; 95 and 4; 95 and 5; 95 and 6; 95 and 7; 95 and 8; 95 and 9;

95 and 10; 95 and 11; 95 and 12; 95 and 13; 95 and 14; 95 and 15; 95 and 16; 95 and 17; 95 and 18; 95 and 19; 95 and 20; 95 and 21; 95 and 22; 95 and 23; 95 and 24; 95 and 25; 95 and 26; 95 and 27; 95 and 28; 95 and 29; 95 and 30; 95 and 31; 95 and 32; 95 and 33; 95 and 34; 95 and 35; 95 and 36; 95 and 37; 95 and 38; 95 and 39; 95 and 40; 95 and 41; 95 and 42; 95 and 43; 95 and 44; 95 and 45; 95 and 46; 95 and 47; 95 and 48; 95 and 49; 95 and 50; 95 and 51; 95 and 52; 95 and 53; 95 and 54; 95 and 55;

95 and 56; 95 and 57; 95 and 58; 95 and 59; 95 and 60; 95 and 61; 95 and 62; 95 and 63; 95 and 64; 96 and 1; 96 and 2; 96 and 3; 96 and 4; 96 and 5; 96 and 6; 96 and 7;

96 and 8; 96 and 9; 96 and 10; 96 and 11; 96 and 12; 96 and 13; 96 and 14; 96 and 15; 96 and 16; 96 and 17; 96 and 18; 96 and 19; 96 and 20; 96 and 21; 96 and 22; 96 and 23; 96 and 24; 96 and 25; 96 and 26; 96 and 27; 96 and 28; 96 and 29; 96 and 30; 96 and 31; 96 and 32; 96 and 33; 96 and 34; 96 and 35; 96 and 36; 96 and 37; 96 and 38; 96 and 39; 96 and 40; 96 and 41; 96 and 42; 96 and 43; 96 and 44; 96 and 45; 96 and 46; 96 and 47; 96 and 48; 96 and 49; 96 and 50; 96 and 51; 96 and 52; 96 and 53;

96 and 54; 96 and 55; 96 and 56; 96 and 57; 96 and 58; 96 and 59; 96 and 60; 96 and 61; 96 and 62; 96 and 63; 96 and 64; 97 and 1; 97 and 2; 97 and 3; 97 and 4; 97 and 5; 97 and 6; 97 and 7; 97 and 8; 97 and 9; 97 and 10; 97 and 11; 97 and 12; 97 and 13; 97 and 14; 97 and 15; 97 and 16; 97 and 17; 97 and 18; 97 and 19; 97 and 20; 97 and 21; 97 and 22; 97 and 23; 97 and 24; 97 and 25; 97 and 26; 97 and 27; 97 and 28;

97 and 29; 97 and 30; 97 and 31; 97 and 32; 97 and 33; 97 and 34; 97 and 35; 97 and 36; 97 and 37; 97 and 38; 97 and 39; 97 and 40; 97 and 41; 97 and 42; 97 and 43; 97 and 44; 97 and 45; 97 and 46; 97 and 47; 97 and 48; 97 and 49; 97 and 50; 97 and 51;

97 and 52; 97 and 53; 97 and 54; 97 and 55; 97 and 56; 97 and 57; 97 and 58; 97 and 59; 97 and 60; 97 and 61; 97 and 62; 97 and 63; 97 and 64; 98 and 1; 98 and 2; 98 and 3; 98 and 4; 98 and 5; 98 and 6; 98 and 7; 98 and 8; 98 and 9; 98 and 10; 98 and 11; 98 and 12; 98 and 13; 98 and 14; 98 and 15; 98 and 16; 98 and 17; 98 and 18; 98 and 19; 98 and 20; 98 and 21; 98 and 22; 98 and 23; 98 and 24; 98 and 25; 98 and 26;

98 and 27; 98 and 28; 98 and 29; 98 and 30; 98 and 31; 98 and 32; 98 and 33; 98 and 34; 98 and 35; 98 and 36; 98 and 37; 98 and 38; 98 and 39; 98 and 40; 98 and 41; 98 and 42; 98 and 43; 98 and 44; 98 and 45; 98 and 46; 98 and 47; 98 and 48; 98 and 49; 98 and 50; 98 and 51; 98 and 52; 98 and 53; 98 and 54; 98 and 55; 98 and 56; 98 and 57; 98 and 58; 98 and 59; 98 and 60; 98 and 61; 98 and 62; 98 and 63; 98 and 64; 99 and 1; 99 and 2; 99 and 3; 99 and 4; 99 and 5; 99 and 6; 99 and 7; 99 and 8; 99 and 9; 99 and 10; 99 and 11; 99 and 12; 99 and 13; 99 and 14; 99 and 15; 99 and 16; 99 and 17; 99 and 18; 99 and 19; 99 and 20; 99 and 21; 99 and 22; 99 and 23; 99 and 24; 99 and 25; 99 and 26; 99 and 27; 99 and 28; 99 and 29; 99 and 30; 99 and 31; 99 and 32; 99 and 33; 99 and 34; 99 and 35; 99 and 36; 99 and 37; 99 and 38; 99 and 39; 99 and 40; 99 and 41; 99 and 42; 99 and 43; 99 and 44; 99 and 45; 99 and 46; 99 and 47; 99 and 48; 99 and 49; 99 and 50; 99 and 51; 99 and 52; 99 and 53; 99 and 54; 99 and 55;

99 and 56; 99 and 57; 99 and 58; 99 and 59; 99 and 60; 99 and 61; 99 and 62; 99 and

63; 99 and 64; 100 and 1; 100 and 2; 100 and 3; 100 and 4; 100 and 5; 100 and 6; 100 and 7; 100 and 8; 100 and 9; 100 and 10; 100 and 11; 100 and 12; 100 and 13; 100 and 14; 100 and 15; 100 and 16; 100 and 17; 100 and 18; 100 and 19; 100 and 20;

100 and 21; 100 and 22; 100 and 23; 100 and 24; 100 and 25; 100 and 26; 100 and

27; 100 and 28; 100 and 29; 100 and 30; 100 and 31; 100 and 32; 100 and 33; 100 and 34; 100 and 35; 100 and 36; 100 and 37; 100 and 38; 100 and 39; 100 and 40;

100 and 41; 100 and 42; 100 and 43; 100 and 44; 100 and 45; 100 and 46; 100 and

47; 100 and 48; 100 and 49; 100 and 50; 100 and 51; 100 and 52; 100 and 53; 100 and 54; 100 and 55; 100 and 56; 100 and 57; 100 and 58; 100 and 59; 100 and 60;

100 and 61; 100 and 62; 100 and 63; 100 and 64; 101 and 1; 101 and 2; 101 and 3;

101 and 4; 101 and 5; 101 and 6; 101 and 7; 101 and 8; 101 and 9; 101 and 10; 101 and 11; 101 and 12; 101 and 13; 101 and 14; 101 and 15; 101 and 16; 101 and 17;

101 and 18; 101 and 19; 101 and 20; 101 and 21; 101 and 22; 101 and 23; 101 and

24; 101 and 25; 101 and 26; 101 and 27; 101 and 28; 101 and 29; 101 and 30; 101 and 31; 101 and 32; 101 and 33; 101 and 34; 101 and 35; 101 and 36; 101 and 37;

101 and 38; 101 and 39; 101 and 40; 101 and 41; 101 and 42; 101 and 43; 101 and

44; 101 and 45; 101 and 46; 101 and 47; 101 and 48; 101 and 49; 101 and 50; 101 and 51; 101 and 52; 101 and 53; 101 and 54; 101 and 55; 101 and 56; 101 and 57;

101 and 58; 101 and 59; 101 and 60; 101 and 61; 101 and 62; 101 and 63; 101 and

64; 102 and 1; 102 and 2; 102 and 3; 102 and 4; 102 and 5; 102 and 6; 102 and 7; 102 and 8; 102 and 9; 102 and 10; 102 and 11; 102 and 12; 102 and 13; 102 and 14; 102 and 15; 102 and 16; 102 and 17; 102 and 18; 102 and 19; 102 and 20; 102 and 21;

102 and 22; 102 and 23; 102 and 24; 102 and 25; 102 and 26; 102 and 27; 102 and

28; 102 and 29; 102 and 30; 102 and 31; 102 and 32; 102 and 33; 102 and 34; 102 and 35; 102 and 36; 102 and 37; 102 and 38; 102 and 39; 102 and 40; 102 and 41;

102 and 42; 102 and 43; 102 and 44; 102 and 45; 102 and 46; 102 and 47; 102 and

48; 102 and 49; 102 and 50; 102 and 51; 102 and 52; 102 and 53; 102 and 54; 102 and 55; 102 and 56; 102 and 57; 102 and 58; 102 and 59; 102 and 60; 102 and 61;

102 and 62; 102 and 63; 102 and 64; 103 and 1; 103 and 2; 103 and 3; 103 and 4; 103 and 5; 103 and 6; 103 and 7; 103 and 8; 103 and 9; 103 and 10; 103 and 11; 103 and 12; 103 and 13; 103 and 14; 103 and 15; 103 and 16; 103 and 17; 103 and 18; 103 and 19; 103 and 20; 103 and 21; 103 and 22; 103 and 23; 103 and 24; 103 and 25;

103 and 26; 103 and 27; 103 and 28; 103 and 29; 103 and 30; 103 and 31; 103 and

32; 103 and 33; 103 and 34; 103 and 35; 103 and 36; 103 and 37; 103 and 38; 103 and 39; 103 and 40; 103 and 41; 103 and 42; 103 and 43; 103 and 44; 103 and 45;

103 and 46; 103 and 47; 103 and 48; 103 and 49; 103 and 50; 103 and 51; 103 and

52; 103 and 53; 103 and 54; 103 and 55; 103 and 56; 103 and 57; 103 and 58; 103 and 59; 103 and 60; 103 and 61; 103 and 62; 103 and 63; 103 and 64; 104 and 1; 104 and 2; 104 and 3; 104 and 4; 104 and 5; 104 and 6; 104 and 7; 104 and 8; 104 and 9;

104 and 10; 104 and 11; 104 and 12; 104 and 13; 104 and 14; 104 and 15; 104 and

16; 104 and 17; 104 and 18; 104 and 19; 104 and 20; 104 and 21; 104 and 22; 104 and 23; 104 and 24; 104 and 25; 104 and 26; 104 and 27; 104 and 28; 104 and 29;

104 and 30; 104 and 31; 104 and 32; 104 and 33; 104 and 34; 104 and 35; 104 and

36; 104 and 37; 104 and 38; 104 and 39; 104 and 40; 104 and 41; 104 and 42; 104 and 43; 104 and 44; 104 and 45; 104 and 46; 104 and 47; 104 and 48; 104 and 49;

104 and 50; 104 and 51; 104 and 52; 104 and 53; 104 and 54; 104 and 55; 104 and

56; 104 and 57; 104 and 58; 104 and 59; 104 and 60; 104 and 61; 104 and 62; 104 and 63; 104 and 64; 105 and 1; 105 and 2; 105 and 3; 105 and 4; 105 and 5; 105 and 6; 105 and 7; 105 and 8; 105 and 9; 105 and 10; 105 and 11; 105 and 12; 105 and 13;

105 and 14; 105 and 15; 105 and 16; 105 and 17; 105 and 18; 105 and 19; 105 and

20; 105 and 21; 105 and 22; 105 and 23; 105 and 24; 105 and 25; 105 and 26; 105 and 27; 105 and 28; 105 and 29; 105 and 30; 105 and 31; 105 and 32; 105 and 33;

105 and 34; 105 and 35; 105 and 36; 105 and 37; 105 and 38; 105 and 39; 105 and

40; 105 and 41; 105 and 42; 105 and 43; 105 and 44; 105 and 45; 105 and 46; 105 and 47; 105 and 48; 105 and 49; 105 and 50; 105 and 51; 105 and 52; 105 and 53;

105 and 54; 105 and 55; 105 and 56; 105 and 57; 105 and 58; 105 and 59; 105 and

60; 105 and 61; 105 and 62; 105 and 63; 105 and 64; 106 and 1; 106 and 2; 106 and 3; 106 and 4; 106 and 5; 106 and 6; 106 and 7; 106 and 8; 106 and 9; 106 and 10; 106 and 11; 106 and 12; 106 and 13; 106 and 14; 106 and 15; 106 and 16; 106 and 17;

106 and 18; 106 and 19; 106 and 20; 106 and 21; 106 and 22; 106 and 23; 106 and

24; 106 and 25; 106 and 26; 106 and 27; 106 and 28; 106 and 29; 106 and 30; 106 and 31; 106 and 32; 106 and 33; 106 and 34; 106 and 35; 106 and 36; 106 and 37;

106 and 38; 106 and 39; 106 and 40; 106 and 41; 106 and 42; 106 and 43; 106 and

44; 106 and 45; 106 and 46; 106 and 47; 106 and 48; 106 and 49; 106 and 50; 106 and 51; 106 and 52; 106 and 53; 106 and 54; 106 and 55; 106 and 56; 106 and 57;

106 and 58; 106 and 59; 106 and 60; 106 and 61; 106 and 62; 106 and 63; 106 and 64; 107 and 1; 107 and 2; 107 and 3; 107 and 4; 107 and 5; 107 and 6; 107 and 7; 107 and 8; 107 and 9; 107 and 10; 107 and 11; 107 and 12; 107 and 13; 107 and 14; 107 and 15; 107 and 16; 107 and 17; 107 and 18; 107 and 19; 107 and 20; 107 and 21;

107 and 22; 107 and 23; 107 and 24; 107 and 25; 107 and 26; 107 and 27; 107 and

28; 107 and 29; 107 and 30; 107 and 31; 107 and 32; 107 and 33; 107 and 34; 107 and 35; 107 and 36; 107 and 37; 107 and 38; 107 and 39; 107 and 40; 107 and 41;

107 and 42; 107 and 43; 107 and 44; 107 and 45; 107 and 46; 107 and 47; 107 and

48; 107 and 49; 107 and 50; 107 and 51; 107 and 52; 107 and 53; 107 and 54; 107 and 55; 107 and 56; 107 and 57; 107 and 58; 107 and 59; 107 and 60; 107 and 61;

107 and 62; 107 and 63; 107 and 64; 108 and 1; 108 and 2; 108 and 3; 108 and 4; 108 and 5; 108 and 6; 108 and 7; 108 and 8; 108 and 9; 108 and 10; 108 and 11; 108 and 12; 108 and 13; 108 and 14; 108 and 15; 108 and 16; 108 and 17; 108 and 18; 108 and 19; 108 and 20; 108 and 21; 108 and 22; 108 and 23; 108 and 24; 108 and 25;

108 and 26; 108 and 27; 108 and 28; 108 and 29; 108 and 30; 108 and 31; 108 and

32; 108 and 33; 108 and 34; 108 and 35; 108 and 36; 108 and 37; 108 and 38; 108 and 39; 108 and 40; 108 and 41; 108 and 42; 108 and 43; 108 and 44; 108 and 45;

108 and 46; 108 and 47; 108 and 48; 108 and 49; 108 and 50; 108 and 51; 108 and

52; 108 and 53; 108 and 54; 108 and 55; 108 and 56; 108 and 57; 108 and 58; 108 and 59; 108 and 60; 108 and 61; 108 and 62; 108 and 63; 108 and 64; 109 and 1; 109 and 2; 109 and 3; 109 and 4; 109 and 5; 109 and 6; 109 and 7; 109 and 8; 109 and 9;

109 and 10; 109 and 11; 109 and 12; 109 and 13; 109 and 14; 109 and 15; 109 and

16; 109 and 17; 109 and 18; 109 and 19; 109 and 20; 109 and 21; 109 and 22; 109 and 23; 109 and 24; 109 and 25; 109 and 26; 109 and 27; 109 and 28; 109 and 29;

109 and 30; 109 and 31; 109 and 32; 109 and 33; 109 and 34; 109 and 35; 109 and

36; 109 and 37; 109 and 38; 109 and 39; 109 and 40; 109 and 41; 109 and 42; 109 and 43; 109 and 44; 109 and 45; 109 and 46; 109 and 47; 109 and 48; 109 and 49;

109 and 50; 109 and 51; 109 and 52; 109 and 53; 109 and 54; 109 and 55; 109 and

56; 109 and 57; 109 and 58; 109 and 59; 109 and 60; 109 and 61; 109 and 62; 109 and 63; 109 and 64; 110 and 1; 110 and 2; 110 and 3; 110 and 4; 110 and 5; 110 and 6; 110 and 7; 110 and 8; 110 and 9; 110 and 10; 110 and 11; 110 and 12; 110 and 13;

110 and 14; 110 and 15; 110 and 16; 110 and 17; 110 and 18; 110 and 19; 110 and

20; 110 and 21; 110 and 22; 110 and 23; 110 and 24; 110 and 25; 110 and 26; 110 and 27; 110 and 28; 110 and 29; 110 and 30; 110 and 31; 110 and 32; 110 and 33;

110 and 34; 110 and 35; 110 and 36; 110 and 37; 110 and 38; 110 and 39; 110 and

40; 110 and 41; 110 and 42; 110 and 43; 110 and 44; 110 and 45; 110 and 46; 110 and 47; 110 and 48; 110 and 49; 110 and 50; 110 and 51; 110 and 52; 110 and 53;

110 and 54; 110 and 55; 110 and 56; 110 and 57; 110 and 58; 110 and 59; 110 and 60; 110 and 61; 110 and 62; 110 and 63; 110 and 64; 111 and 1; 111 and 2; 111 and 3; 111 and 4; 111 and 5; 111 and 6; 111 and 7; 111 and 8; 111 and 9; 111 and 10; 111 and 11; 111 and 12; 111 and 13; 111 and 14; 111 and 15; 111 and 16; 111 and 17;

111 and 18; 111 and 19; 111 and 20; 111 and 21; 111 and 22; 111 and 23; 111 and

24; 111 and 25; 111 and 26; 111 and 27; 111 and 28; 111 and 29; 111 and 30; 111 and 31; 111 and 32; 111 and 33; 111 and 34; 111 and 35; 111 and 36; 111 and 37;

111 and 38; 111 and 39; 111 and 40; 111 and 41; 111 and 42; 111 and 43; 111 and

44; 111 and 45; 111 and 46; 111 and 47; 111 and 48; 111 and 49; 111 and 50; 111 and 51; 111 and 52; 111 and 53; 111 and 54; 111 and 55; 111 and 56; 111 and 57;

111 and 58; 111 and 59; 111 and 60; 111 and 61; 111 and 62; 111 and 63; 111 and

64; 112 and 1; 112 and 2; 112 and 3; 112 and 4; 112 and 5; 112 and 6; 112 and 7; 112 and 8; 112 and 9; 112 and 10; 112 and 11; 112 and 12; 112 and 13; 112 and 14; 112 and 15; 112 and 16; 112 and 17; 112 and 18; 112 and 19; 112 and 20; 112 and 21;

112 and 22; 112 and 23; 112 and 24; 112 and 25; 112 and 26; 112 and 27; 112 and

28; 112 and 29; 112 and 30; 112 and 31; 112 and 32; 112 and 33; 112 and 34; 112 and 35; 112 and 36; 112 and 37; 112 and 38; 112 and 39; 112 and 40; 112 and 41;

112 and 42; 112 and 43; 112 and 44; 112 and 45; 112 and 46; 112 and 47; 112 and

48; 112 and 49; 112 and 50; 112 and 51; 112 and 52; 112 and 53; 112 and 54; 112 and 55; 112 and 56; 112 and 57; 112 and 58; 112 and 59; 112 and 60; 112 and 61;

112 and 62; 112 and 63; 112 and 64; 113 and 1; 113 and 2; 113 and 3; 113 and 4; 113 and 5; 113 and 6; 113 and 7; 113 and 8; 113 and 9; 113 and 10; 113 and 11; 113 and 12; 113 and 13; 113 and 14; 113 and 15; 113 and 16; 113 and 17; 113 and 18; 113 and 19; 113 and 20; 113 and 21; 113 and 22; 113 and 23; 113 and 24; 113 and 25;

113 and 26; 113 and 27; 113 and 28; 113 and 29; 113 and 30; 113 and 31; 113 and

32; 113 and 33; 113 and 34; 113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 43; 113 and 44; 113 and 45;

113 and 46; 113 and 47; 113 and 48; 113 and 49; 113 and 50; 113 and 51; 113 and

52; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 57; 113 and 58; 113 and 59; 113 and 60; 113 and 61; 113 and 62; 113 and 63; 113 and 64; 114 and 1; 114 and 2; 114 and 3; 114 and 4; 114 and 5; 114 and 6; 114 and 7; 114 and 8; 114 and 9;

114 and 10; 114 and 11; 114 and 12; 114 and 13; 114 and 14; 114 and 15; 114 and

16; 114 and 17; 114 and 18; 114 and 19; 114 and 20; 114 and 21; 114 and 22; 114 and 23; 114 and 24; 114 and 25; 114 and 26; 114 and 27; 114 and 28; 114 and 29;

114 and 30; 114 and 31; 114 and 32; 114 and 33; 114 and 34; 114 and 35; 114 and

36; 114 and 37; 114 and 38; 114 and 39; 114 and 40; 114 and 41; 114 and 42; 114 and 43; 114 and 44; 114 and 45; 114 and 46; 114 and 47; 114 and 48; 114 and 49;

114 and 50; 114 and 51; 114 and 52; 114 and 53; 114 and 54; 114 and 55; 114 and 56; 114 and 57; 114 and 58; 114 and 59; 114 and 60; 114 and 61; 114 and 62; 114 and 63; 114 and 64; 115 and 1; 115 and 2; 115 and 3; 115 and 4; 115 and 5; 115 and 6; 115 and 7; 115 and 8; 115 and 9; 115 and 10; 115 and 11; 115 and 12; 115 and 13; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 18; 115 and 19; 115 and

20; 115 and 21; 115 and 22; 115 and 23; 115 and 24; 115 and 25; 115 and 26; 115 and 27; 115 and 28; 115 and 29; 115 and 30; 115 and 31; 115 and 32; 115 and 33;

115 and 34; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and

40; 115 and 41; 115 and 42; 115 and 43; 115 and 44; 115 and 45; 115 and 46; 115 and 47; 115 and 48; 115 and 49; 115 and 50; 115 and 51; 115 and 52; 115 and 53;

115 and 54; 115 and 55; 115 and 56; 115 and 57; 115 and 58; 115 and 59; 115 and

60; 115 and 61; 115 and 62; 115 and 63; 115 and 64; 116 and 1; 116 and 2; 116 and 3; 116 and 4; 116 and 5; 116 and 6; 116 and 7; 116 and 8; 116 and 9; 116 and 10; 116 and 11; 116 and 12; 116 and 13; 116 and 14; 116 and 15; 116 and 16; 116 and 17;

116 and 18; 116 and 19; 116 and 20; 116 and 21; 116 and 22; 116 and 23; 116 and

24; 116 and 25; 116 and 26; 116 and 27; 116 and 28; 116 and 29; 116 and 30; 116 and 31; 116 and 32; 116 and 33; 116 and 34; 116 and 35; 116 and 36; 116 and 37;

116 and 38; 116 and 39; 116 and 40; 116 and 41; 116 and 42; 116 and 43; 116 and

44; 116 and 45; 116 and 46; 116 and 47; 116 and 48; 116 and 49; 116 and 50; 116 and 51; 116 and 52; 116 and 53; 116 and 54; 116 and 55; 116 and 56; 116 and 57;

116 and 58; 116 and 59; 116 and 60; 116 and 61; 116 and 62; 116 and 63; 116 and

64; 117 and 1; 117 and 2; 117 and 3; 117 and 4; 117 and 5; 117 and 6; 117 and 7; 117 and 8; 117 and 9; 117 and 10; 117 and 11; 117 and 12; 117 and 13; 117 and 14; 117 and 15; 117 and 16; 117 and 17; 117 and 18; 117 and 19; 117 and 20; 117 and 21;

117 and 22; 117 and 23; 117 and 24; 117 and 25; 117 and 26; 117 and 27; 117 and

28; 117 and 29; 117 and 30; 117 and 31; 117 and 32; 117 and 33; 117 and 34; 117 and 35; 117 and 36; 117 and 37; 117 and 38; 117 and 39; 117 and 40; 117 and 41;

117 and 42; 117 and 43; 117 and 44; 117 and 45; 117 and 46; 117 and 47; 117 and

48; 117 and 49; 117 and 50; 117 and 51; 117 and 52; 117 and 53; 117 and 54; 117 and 55; 117 and 56; 117 and 57; 117 and 58; 117 and 59; 117 and 60; 117 and 61;

117 and 62; 117 and 63; 117 and 64; 118 and 1; 118 and 2; 118 and 3; 118 and 4; 118 and 5; 118 and 6; 118 and 7; 118 and 8; 118 and 9; 118 and 10; 118 and 11; 118 and 12; 118 and 13; 118 and 14; 118 and 15; 118 and 16; 118 and 17; 118 and 18; 118 and 19; 118 and 20; 118 and 21; 118 and 22; 118 and 23; 118 and 24; 118 and 25;

118 and 26; 118 and 27; 118 and 28; 118 and 29; 118 and 30; 118 and 31; 118 and

32; 118 and 33; 118 and 34; 118 and 35; 118 and 36; 118 and 37; 118 and 38; 118 and 39; 118 and 40; 118 and 41; 118 and 42; 118 and 43; 118 and 44; 118 and 45;

118 and 46; 118 and 47; 118 and 48; 118 and 49; 118 and 50; 118 and 51; 118 and 52; 118 and 53; 118 and 54; 118 and 55; 118 and 56; 118 and 57; 118 and 58; 118 and 59; 118 and 60; 118 and 61; 118 and 62; 118 and 63; 118 and 64; 119 and 1; 119 and 2; 119 and 3; 119 and 4; 119 and 5; 119 and 6; 119 and 7; 119 and 8; 119 and 9; 119 and 10; 119 and 11; 119 and 12; 119 and 13; 119 and 14; 119 and 15; 119 and

16; 119 and 17; 119 and 18; 119 and 19; 119 and 20; 119 and 21; 119 and 22; 119 and 23; 119 and 24; 119 and 25; 119 and 26; 119 and 27; 119 and 28; 119 and 29;

119 and 30; 119 and 31; 119 and 32; 119 and 33; 119 and 34; 119 and 35; 119 and

36; 119 and 37; 119 and 38; 119 and 39; 119 and 40; 119 and 41; 119 and 42; 119 and 43; 119 and 44; 119 and 45; 119 and 46; 119 and 47; 119 and 48; 119 and 49;

119 and 50; 119 and 51; 119 and 52; 119 and 53; 119 and 54; 119 and 55; 119 and

56; 119 and 57; 119 and 58; 119 and 59; 119 and 60; 119 and 61; 119 and 62; 119 and 63; 119 and 64; 120 and 1; 120 and 2; 120 and 3; 120 and 4; 120 and 5; 120 and 6; 120 and 7; 120 and 8; 120 and 9; 120 and 10; 120 and 11; 120 and 12; 120 and 13;

120 and 14; 120 and 15; 120 and 16; 120 and 17; 120 and 18; 120 and 19; 120 and

20; 120 and 21; 120 and 22; 120 and 23; 120 and 24; 120 and 25; 120 and 26; 120 and 27; 120 and 28; 120 and 29; 120 and 30; 120 and 31; 120 and 32; 120 and 33;

120 and 34; 120 and 35; 120 and 36; 120 and 37; 120 and 38; 120 and 39; 120 and

40; 120 and 41; 120 and 42; 120 and 43; 120 and 44; 120 and 45; 120 and 46; 120 and 47; 120 and 48; 120 and 49; 120 and 50; 120 and 51; 120 and 52; 120 and 53;

120 and 54; 120 and 55; 120 and 56; 120 and 57; 120 and 58; 120 and 59; 120 and

60; 120 and 61; 120 and 62; 120 and 63; 120 and 64; 121 and 1; 121 and 2; 121 and 3; 121 and 4; 121 and 5; 121 and 6; 121 and 7; 121 and 8; 121 and 9; 121 and 10; 121 and 11; 121 and 12; 121 and 13; 121 and 14; 121 and 15; 121 and 16; 121 and 17;

121 and 18; 121 and 19; 121 and 20; 121 and 21; 121 and 22; 121 and 23; 121 and

24; 121 and 25; 121 and 26; 121 and 27; 121 and 28; 121 and 29; 121 and 30; 121 and 31; 121 and 32; 121 and 33; 121 and 34; 121 and 35; 121 and 36; 121 and 37;

121 and 38; 121 and 39; 121 and 40; 121 and 41; 121 and 42; 121 and 43; 121 and

44; 121 and 45; 121 and 46; 121 and 47; 121 and 48; 121 and 49; 121 and 50; 121 and 51; 121 and 52; 121 and 53; 121 and 54; 121 and 55; 121 and 56; 121 and 57;

121 and 58; 121 and 59; 121 and 60; 121 and 61; 121 and 62; 121 and 63; 121 and

64; 122 and 1; 122 and 2; 122 and 3; 122 and 4; 122 and 5; 122 and 6; 122 and 7; 122 and 8; 122 and 9; 122 and 10; 122 and 11; 122 and 12; 122 and 13; 122 and 14; 122 and 15; 122 and 16; 122 and 17; 122 and 18; 122 and 19; 122 and 20; 122 and 21;

122 and 22; 122 and 23; 122 and 24; 122 and 25; 122 and 26; 122 and 27; 122 and

28; 122 and 29; 122 and 30; 122 and 31; 122 and 32; 122 and 33; 122 and 34; 122 and 35; 122 and 36; 122 and 37; 122 and 38; 122 and 39; 122 and 40; 122 and 41;

122 and 42; 122 and 43; 122 and 44; 122 and 45; 122 and 46; 122 and 47; 122 and 48; 122 and 49; 122 and 50; 122 and 51; 122 and 52; 122 and 53; 122 and 54; 122 and 55; 122 and 56; 122 and 57; 122 and 58; 122 and 59; 122 and 60; 122 and 61;

122 and 62; 122 and 63; 122 and 64; 123 and 1; 123 and 2; 123 and 3; 123 and 4; 123 and 5; 123 and 6; 123 and 7; 123 and 8; 123 and 9; 123 and 10; 123 and 11; 123 and 12; 123 and 13; 123 and 14; 123 and 15; 123 and 16; 123 and 17; 123 and 18; 123 and 19; 123 and 20; 123 and 21; 123 and 22; 123 and 23; 123 and 24; 123 and 25;

123 and 26; 123 and 27; 123 and 28; 123 and 29; 123 and 30; 123 and 31; 123 and 32; 123 and 33; 123 and 34; 123 and 35; 123 and 36; 123 and 37; 123 and 38; 123 and 39; 123 and 40; 123 and 41; 123 and 42; 123 and 43; 123 and 44; 123 and 45;

123 and 46; 123 and 47; 123 and 48; 123 and 49; 123 and 50; 123 and 51; 123 and 52; 123 and 53; 123 and 54; 123 and 55; 123 and 56; 123 and 57; 123 and 58; 123 and 59; 123 and 60; 123 and 61; 123 and 62; 123 and 63; 123 and 64; 124 and 1; 124 and 2; 124 and 3; 124 and 4; 124 and 5; 124 and 6; 124 and 7; 124 and 8; 124 and 9;

124 and 10; 124 and 11; 124 and 12; 124 and 13; 124 and 14; 124 and 15; 124 and 16; 124 and 17; 124 and 18; 124 and 19; 124 and 20; 124 and 21; 124 and 22; 124 and 23; 124 and 24; 124 and 25; 124 and 26; 124 and 27; 124 and 28; 124 and 29; 124 and 30; 124 and 31; 124 and 32; 124 and 33; 124 and 34; 124 and 35; 124 and 36; 124 and 37; 124 and 38; 124 and 39; 124 and 40; 124 and 41; 124 and 42; 124 and 43; 124 and 44; 124 and 45; 124 and 46; 124 and 47; 124 and 48; 124 and 49;

124 and 50; 124 and 51; 124 and 52; 124 and 53; 124 and 54; 124 and 55; 124 and 56; 124 and 57; 124 and 58; 124 and 59; 124 and 60; 124 and 61; 124 and 62; 124 and 63; 124 and 64; 125 and 1; 125 and 2; 125 and 3; 125 and 4; 125 and 5; 125 and 6; 125 and 7; 125 and 8; 125 and 9; 125 and 10; 125 and 11; 125 and 12; 125 and 13;

125 and 14; 125 and 15; 125 and 16; 125 and 17; 125 and 18; 125 and 19; 125 and 20; 125 and 21; 125 and 22; 125 and 23; 125 and 24; 125 and 25; 125 and 26; 125 and 27; 125 and 28; 125 and 29; 125 and 30; 125 and 31; 125 and 32; 125 and 33; 125 and 34; 125 and 35; 125 and 36; 125 and 37; 125 and 38; 125 and 39; 125 and 40; 125 and 41; 125 and 42; 125 and 43; 125 and 44; 125 and 45; 125 and 46; 125 and 47; 125 and 48; 125 and 49; 125 and 50; 125 and 51; 125 and 52; 125 and 53;

125 and 54; 125 and 55; 125 and 56; 125 and 57; 125 and 58; 125 and 59; 125 and

60; 125 and 61; 125 and 62; 125 and 63; 125 and 64; 126 and 1; 126 and 2; 126 and 3; 126 and 4; 126 and 5; 126 and 6; 126 and 7; 126 and 8; 126 and 9; 126 and 10; 126 and 11; 126 and 12; 126 and 13; 126 and 14; 126 and 15; 126 and 16; 126 and 17;

126 and 18; 126 and 19; 126 and 20; 126 and 21; 126 and 22; 126 and 23; 126 and

24; 126 and 25; 126 and 26; 126 and 27; 126 and 28; 126 and 29; 126 and 30; 126 and 31; 126 and 32; 126 and 33; 126 and 34; 126 and 35; 126 and 36; 126 and 37;

126 and 38; 126 and 39; 126 and 40; 126 and 41; 126 and 42; 126 and 43; 126 and 44; 126 and 45; 126 and 46; 126 and 47; 126 and 48; 126 and 49; 126 and 50; 126 and 51; 126 and 52; 126 and 53; 126 and 54; 126 and 55; 126 and 56; 126 and 57;

126 and 58; 126 and 59; 126 and 60; 126 and 61; 126 and 62; 126 and 63; 126 and

64; 127 and 1; 127 and 2; 127 and 3; 127 and 4; 127 and 5; 127 and 6; 127 and 7; 127 and 8; 127 and 9; 127 and 10; 127 and 11; 127 and 12; 127 and 13; 127 and 14; 127 and 15; 127 and 16; 127 and 17; 127 and 18; 127 and 19; 127 and 20; 127 and 21;

127 and 22; 127 and 23; 127 and 24; 127 and 25; 127 and 26; 127 and 27; 127 and

28; 127 and 29; 127 and 30; 127 and 31; 127 and 32; 127 and 33; 127 and 34; 127 and 35; 127 and 36; 127 and 37; 127 and 38; 127 and 39; 127 and 40; 127 and 41;

127 and 42; 127 and 43; 127 and 44; 127 and 45; 127 and 46; 127 and 47; 127 and

48; 127 and 49; 127 and 50; 127 and 51; 127 and 52; 127 and 53; 127 and 54; 127 and 55; 127 and 56; 127 and 57; 127 and 58; 127 and 59; 127 and 60; 127 and 61;

127 and 62; 127 and 63; 127 and 64; 128 and 1; 128 and 2; 128 and 3; 128 and 4; 128 and 5; 128 and 6; 128 and 7; 128 and 8; 128 and 9; 128 and 10; 128 and 11; 128 and 12; 128 and 13; 128 and 14; 128 and 15; 128 and 16; 128 and 17; 128 and 18; 128 and 19; 128 and 20; 128 and 21; 128 and 22; 128 and 23; 128 and 24; 128 and 25;

128 and 26; 128 and 27; 128 and 28; 128 and 29; 128 and 30; 128 and 31; 128 and

32; 128 and 33; 128 and 34; 128 and 35; 128 and 36; 128 and 37; 128 and 38; 128 and 39; 128 and 40; 128 and 41; 128 and 42; 128 and 43; 128 and 44; 128 and 45;

128 and 46; 128 and 47; 128 and 48; 128 and 49; 128 and 50; 128 and 51; 128 and

52; 128 and 53; 128 and 54; 128 and 55; 128 and 56; 128 and 57; 128 and 58; 128 and 59; 128 and 60; 128 and 61; 128 and 62; 128 and 63; 128 and 64; 129 and 1; 129 and 2; 129 and 3; 129 and 4; 129 and 5; 129 and 6; 129 and 7; 129 and 8; 129 and 9;

129 and 10; 129 and 11; 129 and 12; 129 and 13; 129 and 14; 129 and 15; 129 and

16; 129 and 17; 129 and 18; 129 and 19; 129 and 20; 129 and 21; 129 and 22; 129 and 23; 129 and 24; 129 and 25; 129 and 26; 129 and 27; 129 and 28; 129 and 29;

129 and 30; 129 and 31; 129 and 32; 129 and 33; 129 and 34; 129 and 35; 129 and

36; 129 and 37; 129 and 38; 129 and 39; 129 and 40; 129 and 41; 129 and 42; 129 and 43; 129 and 44; 129 and 45; 129 and 46; 129 and 47; 129 and 48; 129 and 49;

129 and 50; 129 and 51; 129 and 52; 129 and 53; 129 and 54; 129 and 55; 129 and

56; 129 and 57; 129 and 58; 129 and 59; 129 and 60; 129 and 61; 129 and 62; 129 and 63; 129 and 64; 130 and 1; 130 and 2; 130 and 3; 130 and 4; 130 and 5; 130 and 6; 130 and 7; 130 and 8; 130 and 9; 130 and 10; 130 and 11; 130 and 12; 130 and 13;

130 and 14; 130 and 15; 130 and 16; 130 and 17; 130 and 18; 130 and 19; 130 and

20; 130 and 21; 130 and 22; 130 and 23; 130 and 24; 130 and 25; 130 and 26; 130 and 27; 130 and 28; 130 and 29; 130 and 30; 130 and 31; 130 and 32; 130 and 33;

130 and 34; 130 and 35; 130 and 36; 130 and 37; 130 and 38; 130 and 39; 130 and 40; 130 and 41; 130 and 42; 130 and 43; 130 and 44; 130 and 45; 130 and 46; 130 and 47; 130 and 48; 130 and 49; 130 and 50; 130 and 51; 130 and 52; 130 and 53;

130 and 54; 130 and 55; 130 and 56; 130 and 57; 130 and 58; 130 and 59; 130 and

60; 130 and 61; 130 and 62; 130 and 63; 130 and 64; 131 and 1; 131 and 2; 131 and

3; 131 and 4; 131 and 5; 131 and 6; 131 and 7; 131 and 8; 131 and 9; 131 and 10; 131 and 11; 131 and 12; 131 and 13; 131 and 14; 131 and 15; 131 and 16; 131 and 17;

131 and 18; 131 and 19; 131 and 20; 131 and 21; 131 and 22; 131 and 23; 131 and

24; 131 and 25; 131 and 26; 131 and 27; 131 and 28; 131 and 29; 131 and 30; 131 and 31; 131 and 32; 131 and 33; 131 and 34; 131 and 35; 131 and 36; 131 and 37;

131 and 38; 131 and 39; 131 and 40; 131 and 41; 131 and 42; 131 and 43; 131 and

44; 131 and 45; 131 and 46; 131 and 47; 131 and 48; 131 and 49; 131 and 50; 131 and 51; 131 and 52; 131 and 53; 131 and 54; 131 and 55; 131 and 56; 131 and 57;

131 and 58; 131 and 59; 131 and 60; 131 and 61; 131 and 62; 131 and 63; 131 and

64; 132 and 1; 132 and 2; 132 and 3; 132 and 4; 132 and 5; 132 and 6; 132 and 7; 132 and 8; 132 and 9; 132 and 10; 132 and 11; 132 and 12; 132 and 13; 132 and 14; 132 and 15; 132 and 16; 132 and 17; 132 and 18; 132 and 19; 132 and 20; 132 and 21;

132 and 22; 132 and 23; 132 and 24; 132 and 25; 132 and 26; 132 and 27; 132 and

28; 132 and 29; 132 and 30; 132 and 31; 132 and 32; 132 and 33; 132 and 34; 132 and 35; 132 and 36; 132 and 37; 132 and 38; 132 and 39; 132 and 40; 132 and 41;

132 and 42; 132 and 43; 132 and 44; 132 and 45; 132 and 46; 132 and 47; 132 and

48; 132 and 49; 132 and 50; 132 and 51; 132 and 52; 132 and 53; 132 and 54; 132 and 55; 132 and 56; 132 and 57; 132 and 58; 132 and 59; 132 and 60; 132 and 61;

132 and 62; 132 and 63; 132 and 64; 133 and 1; 133 and 2; 133 and 3; 133 and 4; 133 and 5; 133 and 6; 133 and 7; 133 and 8; 133 and 9; 133 and 10; 133 and 11; 133 and 12; 133 and 13; 133 and 14; 133 and 15; 133 and 16; 133 and 17; 133 and 18; 133 and 19; 133 and 20; 133 and 21; 133 and 22; 133 and 23; 133 and 24; 133 and 25;

133 and 26; 133 and 27; 133 and 28; 133 and 29; 133 and 30; 133 and 31; 133 and

32; 133 and 33; 133 and 34; 133 and 35; 133 and 36; 133 and 37; 133 and 38; 133 and 39; 133 and 40; 133 and 41; 133 and 42; 133 and 43; 133 and 44; 133 and 45;

133 and 46; 133 and 47; 133 and 48; 133 and 49; 133 and 50; 133 and 51; 133 and

52; 133 and 53; 133 and 54; 133 and 55; 133 and 56; 133 and 57; 133 and 58; 133 and 59; 133 and 60; 133 and 61; 133 and 62; 133 and 63; 133 and 64; 134 and 1; 134 and 2; 134 and 3; 134 and 4; 134 and 5; 134 and 6; 134 and 7; 134 and 8; 134 and 9;

134 and 10; 134 and 11; 134 and 12; 134 and 13; 134 and 14; 134 and 15; 134 and

16; 134 and 17; 134 and 18; 134 and 19; 134 and 20; 134 and 21; 134 and 22; 134 and 23; 134 and 24; 134 and 25; 134 and 26; 134 and 27; 134 and 28; 134 and 29;

134 and 30; 134 and 31; 134 and 32; 134 and 33; 134 and 34; 134 and 35; 134 and 36; 134 and 37; 134 and 38; 134 and 39; 134 and 40; 134 and 41; 134 and 42; 134 and 43; 134 and 44; 134 and 45; 134 and 46; 134 and 47; 134 and 48; 134 and 49;

134 and 50; 134 and 51; 134 and 52; 134 and 53; 134 and 54; 134 and 55; 134 and

56; 134 and 57; 134 and 58; 134 and 59; 134 and 60; 134 and 61; 134 and 62; 134 and 63; 134 and 64; 135 and 1; 135 and 2; 135 and 3; 135 and 4; 135 and 5; 135 and 6; 135 and 7; 135 and 8; 135 and 9; 135 and 10; 135 and 11; 135 and 12; 135 and 13;

135 and 14; 135 and 15; 135 and 16; 135 and 17; 135 and 18; 135 and 19; 135 and

20; 135 and 21; 135 and 22; 135 and 23; 135 and 24; 135 and 25; 135 and 26; 135 and 27; 135 and 28; 135 and 29; 135 and 30; 135 and 31; 135 and 32; 135 and 33;

135 and 34; 135 and 35; 135 and 36; 135 and 37; 135 and 38; 135 and 39; 135 and

40; 135 and 41; 135 and 42; 135 and 43; 135 and 44; 135 and 45; 135 and 46; 135 and 47; 135 and 48; 135 and 49; 135 and 50; 135 and 51; 135 and 52; 135 and 53;

135 and 54; 135 and 55; 135 and 56; 135 and 57; 135 and 58; 135 and 59; 135 and

60; 135 and 61; 135 and 62; 135 and 63; 135 and 64; 136 and 1; 136 and 2; 136 and 3; 136 and 4; 136 and 5; 136 and 6; 136 and 7; 136 and 8; 136 and 9; 136 and 10; 136 and 11; 136 and 12; 136 and 13; 136 and 14; 136 and 15; 136 and 16; 136 and 17;

136 and 18; 136 and 19; 136 and 20; 136 and 21; 136 and 22; 136 and 23; 136 and

24; 136 and 25; 136 and 26; 136 and 27; 136 and 28; 136 and 29; 136 and 30; 136 and 31; 136 and 32; 136 and 33; 136 and 34; 136 and 35; 136 and 36; 136 and 37;

136 and 38; 136 and 39; 136 and 40; 136 and 41; 136 and 42; 136 and 43; 136 and

44; 136 and 45; 136 and 46; 136 and 47; 136 and 48; 136 and 49; 136 and 50; 136 and 51; 136 and 52; 136 and 53; 136 and 54; 136 and 55; 136 and 56; 136 and 57;

136 and 58; 136 and 59; 136 and 60; 136 and 61; 136 and 62; 136 and 63; 136 and

64; 137 and 1; 137 and 2; 137 and 3; 137 and 4; 137 and 5; 137 and 6; 137 and 7; 137 and 8; 137 and 9; 137 and 10; 137 and 11; 137 and 12; 137 and 13; 137 and 14; 137 and 15; 137 and 16; 137 and 17; 137 and 18; 137 and 19; 137 and 20; 137 and 21;

137 and 22; 137 and 23; 137 and 24; 137 and 25; 137 and 26; 137 and 27; 137 and

28; 137 and 29; 137 and 30; 137 and 31; 137 and 32; 137 and 33; 137 and 34; 137 and 35; 137 and 36; 137 and 37; 137 and 38; 137 and 39; 137 and 40; 137 and 41;

137 and 42; 137 and 43; 137 and 44; 137 and 45; 137 and 46; 137 and 47; 137 and

48; 137 and 49; 137 and 50; 137 and 51; 137 and 52; 137 and 53; 137 and 54; 137 and 55; 137 and 56; 137 and 57; 137 and 58; 137 and 59; 137 and 60; 137 and 61;

137 and 62; 137 and 63; 137 and 64; 138 and 1; 138 and 2; 138 and 3; 138 and 4; 138 and 5; 138 and 6; 138 and 7; 138 and 8; 138 and 9; 138 and 10; 138 and 11; 138 and

12; 138 and 13; 138 and 14; 138 and 15; 138 and 16; 138 and 17; 138 and 18; 138 and 19; 138 and 20; 138 and 21; 138 and 22; 138 and 23; 138 and 24; 138 and 25;

138 and 26; 138 and 27; 138 and 28; 138 and 29; 138 and 30; 138 and 31; 138 and 32; 138 and 33; 138 and 34; 138 and 35; 138 and 36; 138 and 37; 138 and 38; 138 and 39; 138 and 40; 138 and 41; 138 and 42; 138 and 43; 138 and 44; 138 and 45;

138 and 46; 138 and 47; 138 and 48; 138 and 49; 138 and 50; 138 and 51; 138 and

52; 138 and 53; 138 and 54; 138 and 55; 138 and 56; 138 and 57; 138 and 58; 138 and 59; 138 and 60; 138 and 61; 138 and 62; 138 and 63; 138 and 64; 139 and 1; 139 and 2; 139 and 3; 139 and 4; 139 and 5; 139 and 6; 139 and 7; 139 and 8; 139 and 9;

139 and 10; 139 and 11; 139 and 12; 139 and 13; 139 and 14; 139 and 15; 139 and

16; 139 and 17; 139 and 18; 139 and 19; 139 and 20; 139 and 21; 139 and 22; 139 and 23; 139 and 24; 139 and 25; 139 and 26; 139 and 27; 139 and 28; 139 and 29;

139 and 30; 139 and 31; 139 and 32; 139 and 33; 139 and 34; 139 and 35; 139 and

36; 139 and 37; 139 and 38; 139 and 39; 139 and 40; 139 and 41; 139 and 42; 139 and 43; 139 and 44; 139 and 45; 139 and 46; 139 and 47; 139 and 48; 139 and 49;

139 and 50; 139 and 51; 139 and 52; 139 and 53; 139 and 54; 139 and 55; 139 and

56; 139 and 57; 139 and 58; 139 and 59; 139 and 60; 139 and 61; 139 and 62; 139 and 63; 139 and 64; 140 and 1; 140 and 2; 140 and 3; 140 and 4; 140 and 5; 140 and 6; 140 and 7; 140 and 8; 140 and 9; 140 and 10; 140 and 11; 140 and 12; 140 and 13;

140 and 14; 140 and 15; 140 and 16; 140 and 17; 140 and 18; 140 and 19; 140 and

20; 140 and 21; 140 and 22; 140 and 23; 140 and 24; 140 and 25; 140 and 26; 140 and 27; 140 and 28; 140 and 29; 140 and 30; 140 and 31; 140 and 32; 140 and 33;

140 and 34; 140 and 35; 140 and 36; 140 and 37; 140 and 38; 140 and 39; 140 and

40; 140 and 41; 140 and 42; 140 and 43; 140 and 44; 140 and 45; 140 and 46; 140 and 47; 140 and 48; 140 and 49; 140 and 50; 140 and 51; 140 and 52; 140 and 53;

140 and 54; 140 and 55; 140 and 56; 140 and 57; 140 and 58; 140 and 59; 140 and

60; 140 and 61; 140 and 62; 140 and 63; 140 and 64; 141 and 1; 141 and 2; 141 and 3; 141 and 4; 141 and 5; 141 and 6; 141 and 7; 141 and 8; 141 and 9; 141 and 10; 141 and 11; 141 and 12; 141 and 13; 141 and 14; 141 and 15; 141 and 16; 141 and 17;

141 and 18; 141 and 19; 141 and 20; 141 and 21; 141 and 22; 141 and 23; 141 and

24; 141 and 25; 141 and 26; 141 and 27; 141 and 28; 141 and 29; 141 and 30; 141 and 31; 141 and 32; 141 and 33; 141 and 34; 141 and 35; 141 and 36; 141 and 37;

141 and 38; 141 and 39; 141 and 40; 141 and 41; 141 and 42; 141 and 43; 141 and

44; 141 and 45; 141 and 46; 141 and 47; 141 and 48; 141 and 49; 141 and 50; 141 and 51; 141 and 52; 141 and 53; 141 and 54; 141 and 55; 141 and 56; 141 and 57;

141 and 58; 141 and 59; 141 and 60; 141 and 61; 141 and 62; 141 and 63; 141 and

64; 142 and 1; 142 and 2; 142 and 3; 142 and 4; 142 and 5; 142 and 6; 142 and 7; 142 and 8; 142 and 9; 142 and 10; 142 and 11; 142 and 12; 142 and 13; 142 and 14; 142 and 15; 142 and 16; 142 and 17; 142 and 18; 142 and 19; 142 and 20; 142 and 21;

142 and 22; 142 and 23; 142 and 24; 142 and 25; 142 and 26; 142 and 27; 142 and 28; 142 and 29; 142 and 30; 142 and 31; 142 and 32; 142 and 33; 142 and 34; 142 and 35; 142 and 36; 142 and 37; 142 and 38; 142 and 39; 142 and 40; 142 and 41;

142 and 42; 142 and 43; 142 and 44; 142 and 45; 142 and 46; 142 and 47; 142 and

48; 142 and 49; 142 and 50; 142 and 51; 142 and 52; 142 and 53; 142 and 54; 142 and 55; 142 and 56; 142 and 57; 142 and 58; 142 and 59; 142 and 60; 142 and 61;

142 and 62; 142 and 63; 142 and 64; 143 and 1; 143 and 2; 143 and 3; 143 and 4; 143 and 5; 143 and 6; 143 and 7; 143 and 8; 143 and 9; 143 and 10; 143 and 11; 143 and 12; 143 and 13; 143 and 14; 143 and 15; 143 and 16; 143 and 17; 143 and 18; 143 and 19; 143 and 20; 143 and 21; 143 and 22; 143 and 23; 143 and 24; 143 and 25;

143 and 26; 143 and 27; 143 and 28; 143 and 29; 143 and 30; 143 and 31; 143 and

32; 143 and 33; 143 and 34; 143 and 35; 143 and 36; 143 and 37; 143 and 38; 143 and 39; 143 and 40; 143 and 41; 143 and 42; 143 and 43; 143 and 44; 143 and 45;

143 and 46; 143 and 47; 143 and 48; 143 and 49; 143 and 50; 143 and 51; 143 and

52; 143 and 53; 143 and 54; 143 and 55; 143 and 56; 143 and 57; 143 and 58; 143 and 59; 143 and 60; 143 and 61; 143 and 62; 143 and 63; or 143 and 64; or b. SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28; 113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48; 113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and 16; 113 and 17; 113 and 57;

113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and 23; 113 and

24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48; 115 and 13; 115 and 53; 115 and 54; 115 and 55; 115 and 56;

115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18; 115 and

58; 115 and 19; 115 and 20; 115 and 59; 115 and 23; 115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and 26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42.

4. A composition comprising one or more nucleic acid molecules encoding: a. a Staphylococcus lugdunensis Cas9 (SluCas9); and b. at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first guide sequence and a second guide sequence selected from any of the pairs of guide sequences shown in Table ID.

5. A composition comprising: a. a single nucleic acid molecule comprising: i. a nucleic acid encoding Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; or ii. a nucleic acid encoding SluCas9 and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii. a nucleic acid encoding SluCas9 and 1, 2, or 3 guide RNAs; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A or 1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide sequence selected from the pairs of guide sequences shown in Table 1D, and further optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence; or b. two nucleic acid molecules comprising: i. a first nucleic acid encoding SluCas9; and a second nucleic acid that does not encode a SluCas9 and encodes any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; ii. a first nucleic acid encoding SluCas9 and

1. at least one, at least two, or at least three guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. 1, 2, or 3 guide RNAs; and a second nucleic acid that does not encode a SluCas9, optionally wherein the second nucleic acid comprises any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; iii. a first nucleic acid encoding SluCas9 and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SluCas9 and encodes from one to six guide RNAs; or iv. a first nucleic acid encoding SluCas9 and at least two guide RNAs, and a second nucleic acid that does not encode a SluCas9 and encodes at least one additional copy of each of the guide RNAs encoded in the first nucleic acid; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A or 1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from the pairs of guide sequences shown in Table 1D, and optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence.

6. A composition comprising a single nucleic acid molecule encoding at least one pair of guide RNAs and a Cas9, wherein the single nucleic acid molecule comprises: a. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences shown in Table ID; and a second nucleic acid encoding a Staphylococcus lugdunensis Cas9 (SluCas9); or b. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences of: SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28; 113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48; 113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and 16; 113 and 17; 113 and 57; 113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and 23; 113 and 24; 113 and 60; 113 and 62; 113 and 63; 113 and 64;

113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40;

115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48; 115 and 13; 115 and 53; 115 and

54; 115 and 55; 115 and 56; 115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18; 115 and 58; 115 and 19; 115 and 20; 115 and 59; 115 and 23;

115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and

26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38;

92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5;

93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42; and a second nucleic acid encoding a SluCas9.

7. A composition comprising a first and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a Staphylococcus lugdunensis (SluCas9) endonuclease and optionally a first or a first and second guide RNA, and the second nucleic acid molecule comprises a first or a first and second guide RNA, wherein the first guide RNA comprises a first guide sequence and the second guide RNA comprises a second guide sequence selected from any one the following pairs of first and second guide sequences: a. any one of the pairs of first and second guide sequences shown in Table ID; or b. SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28;

113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48; 113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and 16; 113 and 17; 113 and 57;

113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and 23; 113 and

24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and

36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48; 115 and 13; 115 and 53; 115 and 54; 115 and 55; 115 and 56;

115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18; 115 and 58; 115 and 19; 115 and 20; 115 and 59; 115 and 23; 115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and 26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3; 122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42.

8. A composition comprising one or more nucleic acid molecules encoding a Staphylococcus lugdunensis Cas9 (SluCas9) and at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide RNA, and wherein the first guide RNA targets a sequence in intron 53 of a DMD gene and the second guide RNA targets a sequence in intron 52 of the DMD gene.

9. The composition of any one of claims 1-8, comprising at least one pair of guide RNAs, wherein the at least one pair of guide RNAs is capable of excising at least a portion of exon 53 of the DMD gene.

10. The composition of any one of claims 1-9, comprising at least one pair of guide RNAs, wherein the first and the second guide RNAs of the at least one pair of guide RNAs each comprise a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a first and a second guide sequence selected from any one of the following pairs of first and second guide sequences: a. any one of the pairs of first and second guide sequences shown in Table ID; or b. SEQ ID NOs: 113 and 1; 113 and 2; 113 and 3; 113 and 5; 113 and 6; 113 and 28;

113 and 35; 113 and 36; 113 and 37; 113 and 38; 113 and 39; 113 and 40; 113 and 41; 113 and 42; 113 and 9; 113 and 44; 113 and 10; 113 and 45; 113 and 46; 113 and 11; 113 and 47; 113 and 12; 113 and 48; 113 and 13; 113 and 53; 113 and 54; 113 and 55; 113 and 56; 113 and 14; 113 and 15; 113 and 16; 113 and 17; 113 and 57;

113 and 18; 113 and 58; 113 and 19; 113 and 20; 113 and 59; 113 and 23; 113 and

24; 113 and 60; 113 and 62; 113 and 63; 113 and 64; 113 and 25; 113 and 26; 115 and 1; 115 and 2; 115 and 3; 115 and 5; 115 and 6; 115 and 28; 115 and 35; 115 and 36; 115 and 37; 115 and 38; 115 and 39; 115 and 40; 115 and 41; 115 and 42; 115 and 9; 115 and 44; 115 and 10; 115 and 45; 115 and 46; 115 and 11; 115 and 47; 115 and 12; 115 and 48; 115 and 13; 115 and 53; 115 and 54; 115 and 55; 115 and 56;

115 and 14; 115 and 15; 115 and 16; 115 and 17; 115 and 57; 115 and 18; 115 and

58; 115 and 19; 115 and 20; 115 and 59; 115 and 23; 115 and 24; 115 and 60; 115 and 62; 115 and 63; 115 and 64; 115 and 25; 115 and 26; 116 and 1; 116 and 2; 116 and 3; 116 and 5; 117 and 1; 117 and 2; 117 and 3; 117 and 5; 118 and 1; 118 and 2; 118 and 3; 118 and 5; 120 and 1; 120 and 2; 120 and 3; 120 and 5; 120 and 6; 121 and 1; 121 and 2; 121 and 3; 121 and 5; 121 and 6; 122 and 1; 122 and 2; 122 and 3;

122 and 5; 122 and 6; 122 and 28; 92 and 1; 92 and 2; 92 and 3; 92 and 5; 92 and 6; 92 and 28; 92 and 35; 92 and 36; 92 and 37; 92 and 38; 92 and 39; 92 and 40; 92 and 41; 92 and 42; 93 and 1; 93 and 2; 93 and 3; 93 and 5; 93 and 6; 93 and 28; 93 and 35; 93 and 36; 93 and 37; 93 and 38; 93 and 39; 93 and 40; 93 and 41; or 93 and 42.

11. The composition of any one of claims 1-10, wherein: i) the first guide RNA comprises the sequence of SEQ ID NO: 115 and the second guide RNA comprises the sequence of SEQ ID NO: 2; ii) the first guide RNA comprises the sequence of SEQ ID NO: 115, and the second guide RNA comprises the sequence of SEQ ID NO: 3; or iii) the first guide RNA comprises the sequence of SEQ ID NO: 113, and the second guide RNA comprises the sequence of SEQ ID NO: 3.

12. A composition comprising at least one pair of guide RNAs, wherein the at least one pair of guide RNAs is capable of excising a fragment of exon 53 of the DMD gene, and wherein i. the fragment comprises a splice donor site, a first guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases upstream of the splice donor site, and a second guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases downstream of the splice donor site; or ii. the fragment comprises a splice acceptor site, a first guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases upstream of the splice acceptor site, and a second guide RNA of the at least one pair of guide RNAs targets a sequence within 200 nucleobases downstream of the splice acceptor site.

13. The composition of claim 12, wherein i. the first guide RNA of the at least one pair of guide RNAs targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases upstream of the splice acceptor site, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases downstream of the splice acceptor site; or ii. the first guide RNA of the at least one pair of guide RNAs targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases upstream of the splice donor site, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleobases downstream of the splice donor site.

14. The composition of claim 12 or claim 13, wherein a. the fragment of the DMD gene comprises a splice donor site and further comprises a portion of intron 53 and an adjoining portion of exon 53 of the DMD gene; or b. the fragment of the DMD gene comprises a splice acceptor site and further comprises a portion of exon 53 and an adjoining portion of intron 52 of the DMD gene.

15. The composition of any one of claims 12-14, wherein a. the first guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 1-200 of SEQ ID NO: 144, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 201-400 of SEQ ID NO: 144; or b. the first guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 1-200 of SEQ ID NO: 145, and the second guide RNA of the at least one pair of guide RNAs targets a sequence within nucleotides 201-400 of SEQ ID NO: 145.

16. The composition of any one of claims 12-15, further comprising a SluCas9 or a nucleic acid encoding a SluCas9.

17. A composition comprising one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs comprise i) a guide sequence of any one of the guide sequences shown in Tables 1A-1C; ii) at least 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence of any one of the guide sequences shown in Tables 1A-1C; iii) a guide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the guide sequences shown in Tables 1A-1C; or iv) a pair of guide RNAs comprising any two of the guide sequence pairs shown in Tables 1 E or 1F; optionally further comprising a SluCas9 or a nucleic acid encoding a SluCas9.

18. A composition comprising a first guide RNA and a second guide RNA, wherein a. the first guide RNA is selected from any one of SEQ ID NOs: 1-64; and the second guide RNA is selected from any one of SEQ ID NOs: 65-87; or b. the first guide RNA is selected from any one of SEQ ID NOs: 65-87; and the second guide RNA is selected from any one of SEQ ID NOs: 88-143.

19. A composition comprising at least one pair of guide RNAs or a nucleic acid encoding at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprise or consist of a first and a second guide sequence selected from any of the following pairs of first and second guide sequences: a. SEQ ID NOs: 1 and 65; 1 and 66; 1 and 67; 1 and 68; 1 and 69; 1 and 70; 1 and 71; 1 and 72; 1 and 73; 1 and 74; 1 and 75; 1 and 76; 1 and 77; 1 and 78; 1 and 79; 1 and 80; 1 and 81; 1 and 82; 1 and 83; 1 and 84; 1 and 85; 1 and 86; 1 and 87; 2 and 65; 2 and 66; 2 and 67; 2 and 68; 2 and 69; 2 and 70; 2 and 71; 2 and 72; 2 and 73; 2 and 74; 2 and 75; 2 and 76; 2 and 77; 2 and 78; 2 and 79; 2 and 80; 2 and 81; 2 and 82; 2 and 83; 2 and 84; 2 and 85; 2 and 86; 2 and 87; 3 and 65; 3 and 66; 3 and 67; 3 and 68; 3 and 69; 3 and 70; 3 and 71; 3 and 72; 3 and 73; 3 and 74; 3 and 75; 3 and 76; 3 and 77; 3 and 78; 3 and 79; 3 and 80; 3 and 81; 3 and 82; 3 and 83; 3 and 84; 3 and 85; 3 and 86; 3 and 87; 4 and 65; 4 and 66; 4 and 67; 4 and 68; 4 and 69; 4 and 70; 4 and 71; 4 and 72; 4 and 73; 4 and 74; 4 and 75; 4 and 76; 4 and 77; 4 and 78; 4 and 79; 4 and 80; 4 and 81; 4 and 82; 4 and 83; 4 and 84; 4 and 85; 4 and 86; 4 and 87; 5 and 65; 5 and 66; 5 and 67; 5 and 68; 5 and 69; 5 and 70; 5 and 71; 5 and 72; 5 and 73; 5 and 74; 5 and 75; 5 and 76; 5 and 77; 5 and 78; 5 and 79; 5 and 80; 5 and 81; 5 and 82; 5 and 83; 5 and 84; 5 and 85; 5 and 86; 5 and 87; 6 and 65; 6 and 66; 6 and 67; 6 and 68; 6 and 69; 6 and 70; 6 and 71; 6 and 72; 6 and 73; 6 and 74; 6 and 75; 6 and 76; 6 and 77; 6 and 78; 6 and 79; 6 and 80; 6 and 81; 6 and 82; 6 and 83; 6 and 84; 6 and 85; 6 and 86; 6 and 87; 7 and 65; 7 and 66; 7 and 67; 7 and 68; 7 and 69; 7 and 70; 7 and 71; 7 and 72; 7 and 73; 7 and 74; 7 and 75; 7 and 76; 7 and 77; 7 and 78; 7 and 79; 7 and 80; 7 and 81; 7 and 82; 7 and 83; 7 and 84; 7 and 85; 7 and 86; 7 and 87; 8 and 65; 8 and 66; 8 and 67; 8 and 68; 8 and 69; 8 and 70; 8 and 71; 8 and 72; 8 and 73; 8 and 74; 8 and 75; 8 and 76; 8 and 77; 8 and 78; 8 and 79; 8 and 80; 8 and 81; 8 and 82; 8 and 83; 8 and 84; 8 and 85; 8 and 86; 8 and 87; 9 and 65; 9 and 66; 9 and 67; 9 and 68; 9 and 69; 9 and 70; 9 and 71; 9 and 72; 9 and 73; 9 and 74; 9 and 75; 9 and 76; 9 and 77; 9 and 78; 9 and 79; 9 and 80; 9 and 81; 9 and 82; 9 and 83; 9 and 84; 9 and 85; 9 and 86; 9 and 87; 10 and 65; 10 and 66; 10 and 67; 10 and 68; 10 and 69; 10 and 70; 10 and 71; 10 and 72; 10 and 73; 10 and 74; 10 and 75; 10 and 76; 10 and 77; 10 and 78; 10 and 79; 10 and 80; 10 and 81; 10 and 82; 10 and 83; 10 and 84; 10 and 85; 10 and 86; 10 and 87; 11 and 65; 11 and 66; 11 and 67; 11 and 68; 11 and 69; 11 and 70; 11 and 71; 11 and 72; 11 and 73; 11 and 74; 11 and 75; 11 and 76; 11 and 77; 11 and 78; 11 and 79; 11 and 80; 11 and 81; 11 and 82; 11 and 83;

11 and 84; 11 and 85; 11 and 86; 11 and 87; 12 and 65; 12 and 66; 12 and 67; 12 and 68; 12 and 69; 12 and 70; 12 and 71; 12 and 72; 12 and 73; 12 and 74; 12 and 75; 12 and 76; 12 and 77; 12 and 78; 12 and 79; 12 and 80; 12 and 81; 12 and 82; 12 and 83;

12 and 84; 12 and 85; 12 and 86; 12 and 87; 13 and 65; 13 and 66; 13 and 67; 13 and 68; 13 and 69; 13 and 70; 13 and 71; 13 and 72; 13 and 73; 13 and 74; 13 and 75; 13 and 76; 13 and 77; 13 and 78; 13 and 79; 13 and 80; 13 and 81; 13 and 82; 13 and 83;

13 and 84; 13 and 85; 13 and 86; 13 and 87; 14 and 65; 14 and 66; 14 and 67; 14 and 68; 14 and 69; 14 and 70; 14 and 71; 14 and 72; 14 and 73; 14 and 74; 14 and 75; 14 and 76; 14 and 77; 14 and 78; 14 and 79; 14 and 80; 14 and 81; 14 and 82; 14 and 83;

14 and 84; 14 and 85; 14 and 86; 14 and 87; 15 and 65; 15 and 66; 15 and 67; 15 and 68; 15 and 69; 15 and 70; 15 and 71; 15 and 72; 15 and 73; 15 and 74; 15 and 75; 15 and 76; 15 and 77; 15 and 78; 15 and 79; 15 and 80; 15 and 81; 15 and 82; 15 and 83;

15 and 84; 15 and 85; 15 and 86; 15 and 87; 16 and 65; 16 and 66; 16 and 67; 16 and 68; 16 and 69; 16 and 70; 16 and 71; 16 and 72; 16 and 73; 16 and 74; 16 and 75; 16 and 76; 16 and 77; 16 and 78; 16 and 79; 16 and 80; 16 and 81; 16 and 82; 16 and 83;

16 and 84; 16 and 85; 16 and 86; 16 and 87; 17 and 65; 17 and 66; 17 and 67; 17 and 68; 17 and 69; 17 and 70; 17 and 71; 17 and 72; 17 and 73; 17 and 74; 17 and 75; 17 and 76; 17 and 77; 17 and 78; 17 and 79; 17 and 80; 17 and 81; 17 and 82; 17 and 83;

17 and 84; 17 and 85; 17 and 86; 17 and 87; 18 and 65; 18 and 66; 18 and 67; 18 and 68; 18 and 69; 18 and 70; 18 and 71; 18 and 72; 18 and 73; 18 and 74; 18 and 75; 18 and 76; 18 and 77; 18 and 78; 18 and 79; 18 and 80; 18 and 81; 18 and 82; 18 and 83;

18 and 84; 18 and 85; 18 and 86; 18 and 87; 19 and 65; 19 and 66; 19 and 67; 19 and 68; 19 and 69; 19 and 70; 19 and 71; 19 and 72; 19 and 73; 19 and 74; 19 and 75; 19 and 76; 19 and 77; 19 and 78; 19 and 79; 19 and 80; 19 and 81; 19 and 82; 19 and 83;

19 and 84; 19 and 85; 19 and 86; 19 and 87; 20 and 65; 20 and 66; 20 and 67; 20 and 68; 20 and 69; 20 and 70; 20 and 71; 20 and 72; 20 and 73; 20 and 74; 20 and 75; 20 and 76; 20 and 77; 20 and 78; 20 and 79; 20 and 80; 20 and 81; 20 and 82; 20 and 83;

20 and 84; 20 and 85; 20 and 86; 20 and 87; 21 and 65; 21 and 66; 21 and 67; 21 and 68; 21 and 69; 21 and 70; 21 and 71; 21 and 72; 21 and 73; 21 and 74; 21 and 75; 21 and 76; 21 and 77; 21 and 78; 21 and 79; 21 and 80; 21 and 81; 21 and 82; 21 and 83;

21 and 84; 21 and 85; 21 and 86; 21 and 87; 22 and 65; 22 and 66; 22 and 67; 22 and 68; 22 and 69; 22 and 70; 22 and 71; 22 and 72; 22 and 73; 22 and 74; 22 and 75; 22 and 76; 22 and 77; 22 and 78; 22 and 79; 22 and 80; 22 and 81; 22 and 82; 22 and 83;

22 and 84; 22 and 85; 22 and 86; 22 and 87; 23 and 65; 23 and 66; 23 and 67; 23 and 68; 23 and 69; 23 and 70; 23 and 71; 23 and 72; 23 and 73; 23 and 74; 23 and 75; 23 and 76; 23 and 77; 23 and 78; 23 and 79; 23 and 80; 23 and 81; 23 and 82; 23 and 83;

23 and 84; 23 and 85; 23 and 86; 23 and 87; 24 and 65; 24 and 66; 24 and 67; 24 and 68; 24 and 69; 24 and 70; 24 and 71; 24 and 72; 24 and 73; 24 and 74; 24 and 75; 24 and 76; 24 and 77; 24 and 78; 24 and 79; 24 and 80; 24 and 81; 24 and 82; 24 and 83;

24 and 84; 24 and 85; 24 and 86; 24 and 87; 25 and 65; 25 and 66; 25 and 67; 25 and 68; 25 and 69; 25 and 70; 25 and 71; 25 and 72; 25 and 73; 25 and 74; 25 and 75; 25 and 76; 25 and 77; 25 and 78; 25 and 79; 25 and 80; 25 and 81; 25 and 82; 25 and 83;

25 and 84; 25 and 85; 25 and 86; 25 and 87; 26 and 65; 26 and 66; 26 and 67; 26 and 68; 26 and 69; 26 and 70; 26 and 71; 26 and 72; 26 and 73; 26 and 74; 26 and 75; 26 and 76; 26 and 77; 26 and 78; 26 and 79; 26 and 80; 26 and 81; 26 and 82; 26 and 83;

26 and 84; 26 and 85; 26 and 86; 26 and 87; 27 and 65; 27 and 66; 27 and 67; 27 and 68; 27 and 69; 27 and 70; 27 and 71; 27 and 72; 27 and 73; 27 and 74; 27 and 75; 27 and 76; 27 and 77; 27 and 78; 27 and 79; 27 and 80; 27 and 81; 27 and 82; 27 and 83;

27 and 84; 27 and 85; 27 and 86; 27 and 87; 28 and 65; 28 and 66; 28 and 67; 28 and 68; 28 and 69; 28 and 70; 28 and 71; 28 and 72; 28 and 73; 28 and 74; 28 and 75; 28 and 76; 28 and 77; 28 and 78; 28 and 79; 28 and 80; 28 and 81; 28 and 82; 28 and 83;

28 and 84; 28 and 85; 28 and 86; 28 and 87; 29 and 65; 29 and 66; 29 and 67; 29 and 68; 29 and 69; 29 and 70; 29 and 71; 29 and 72; 29 and 73; 29 and 74; 29 and 75; 29 and 76; 29 and 77; 29 and 78; 29 and 79; 29 and 80; 29 and 81; 29 and 82; 29 and 83;

29 and 84; 29 and 85; 29 and 86; 29 and 87; 30 and 65; 30 and 66; 30 and 67; 30 and 68; 30 and 69; 30 and 70; 30 and 71; 30 and 72; 30 and 73; 30 and 74; 30 and 75; 30 and 76; 30 and 77; 30 and 78; 30 and 79; 30 and 80; 30 and 81; 30 and 82; 30 and 83;

30 and 84; 30 and 85; 30 and 86; 30 and 87; 31 and 65; 31 and 66; 31 and 67; 31 and 68; 31 and 69; 31 and 70; 31 and 71; 31 and 72; 31 and 73; 31 and 74; 31 and 75; 31 and 76; 31 and 77; 31 and 78; 31 and 79; 31 and 80; 31 and 81; 31 and 82; 31 and 83;

31 and 84; 31 and 85; 31 and 86; 31 and 87; 32 and 65; 32 and 66; 32 and 67; 32 and 68; 32 and 69; 32 and 70; 32 and 71; 32 and 72; 32 and 73; 32 and 74; 32 and 75; 32 and 76; 32 and 77; 32 and 78; 32 and 79; 32 and 80; 32 and 81; 32 and 82; 32 and 83;

32 and 84; 32 and 85; 32 and 86; 32 and 87; 33 and 65; 33 and 66; 33 and 67; 33 and 68; 33 and 69; 33 and 70; 33 and 71; 33 and 72; 33 and 73; 33 and 74; 33 and 75; 33 and 76; 33 and 77; 33 and 78; 33 and 79; 33 and 80; 33 and 81; 33 and 82; 33 and 83;

33 and 84; 33 and 85; 33 and 86; 33 and 87; 34 and 65; 34 and 66; 34 and 67; 34 and 68; 34 and 69; 34 and 70; 34 and 71; 34 and 72; 34 and 73; 34 and 74; 34 and 75; 34 and 76; 34 and 77; 34 and 78; 34 and 79; 34 and 80; 34 and 81; 34 and 82; 34 and 83;

34 and 84; 34 and 85; 34 and 86; 34 and 87; 35 and 65; 35 and 66; 35 and 67; 35 and 68; 35 and 69; 35 and 70; 35 and 71; 35 and 72; 35 and 73; 35 and 74; 35 and 75; 35 and 76; 35 and 77; 35 and 78; 35 and 79; 35 and 80; 35 and 81; 35 and 82; 35 and 83; 35 and 84; 35 and 85; 35 and 86; 35 and 87; 36 and 65; 36 and 66; 36 and 67; 36 and 68; 36 and 69; 36 and 70; 36 and 71; 36 and 72; 36 and 73; 36 and 74; 36 and 75; 36 and 76; 36 and 77; 36 and 78; 36 and 79; 36 and 80; 36 and 81; 36 and 82; 36 and 83;

36 and 84; 36 and 85; 36 and 86; 36 and 87; 37 and 65; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 37 and 70; 37 and 71; 37 and 72; 37 and 73; 37 and 74; 37 and 75; 37 and 76; 37 and 77; 37 and 78; 37 and 79; 37 and 80; 37 and 81; 37 and 82; 37 and 83;

37 and 84; 37 and 85; 37 and 86; 37 and 87; 38 and 65; 38 and 66; 38 and 67; 38 and 68; 38 and 69; 38 and 70; 38 and 71; 38 and 72; 38 and 73; 38 and 74; 38 and 75; 38 and 76; 38 and 77; 38 and 78; 38 and 79; 38 and 80; 38 and 81; 38 and 82; 38 and 83;

38 and 84; 38 and 85; 38 and 86; 38 and 87; 39 and 65; 39 and 66; 39 and 67; 39 and 68; 39 and 69; 39 and 70; 39 and 71; 39 and 72; 39 and 73; 39 and 74; 39 and 75; 39 and 76; 39 and 77; 39 and 78; 39 and 79; 39 and 80; 39 and 81; 39 and 82; 39 and 83;

39 and 84; 39 and 85; 39 and 86; 39 and 87; 40 and 65; 40 and 66; 40 and 67; 40 and 68; 40 and 69; 40 and 70; 40 and 71; 40 and 72; 40 and 73; 40 and 74; 40 and 75; 40 and 76; 40 and 77; 40 and 78; 40 and 79; 40 and 80; 40 and 81; 40 and 82; 40 and 83;

40 and 84; 40 and 85; 40 and 86; 40 and 87; 41 and 65; 41 and 66; 41 and 67; 41 and 68; 41 and 69; 41 and 70; 41 and 71; 41 and 72; 41 and 73; 41 and 74; 41 and 75; 41 and 76; 41 and 77; 41 and 78; 41 and 79; 41 and 80; 41 and 81; 41 and 82; 41 and 83;

41 and 84; 41 and 85; 41 and 86; 41 and 87; 42 and 65; 42 and 66; 42 and 67; 42 and 68; 42 and 69; 42 and 70; 42 and 71; 42 and 72; 42 and 73; 42 and 74; 42 and 75; 42 and 76; 42 and 77; 42 and 78; 42 and 79; 42 and 80; 42 and 81; 42 and 82; 42 and 83;

42 and 84; 42 and 85; 42 and 86; 42 and 87; 43 and 65; 43 and 66; 43 and 67; 43 and 68; 43 and 69; 43 and 70; 43 and 71; 43 and 72; 43 and 73; 43 and 74; 43 and 75; 43 and 76; 43 and 77; 43 and 78; 43 and 79; 43 and 80; 43 and 81; 43 and 82; 43 and 83;

43 and 84; 43 and 85; 43 and 86; 43 and 87; 44 and 65; 44 and 66; 44 and 67; 44 and 68; 44 and 69; 44 and 70; 44 and 71; 44 and 72; 44 and 73; 44 and 74; 44 and 75; 44 and 76; 44 and 77; 44 and 78; 44 and 79; 44 and 80; 44 and 81; 44 and 82; 44 and 83;

44 and 84; 44 and 85; 44 and 86; 44 and 87; 45 and 65; 45 and 66; 45 and 67; 45 and 68; 45 and 69; 45 and 70; 45 and 71; 45 and 72; 45 and 73; 45 and 74; 45 and 75; 45 and 76; 45 and 77; 45 and 78; 45 and 79; 45 and 80; 45 and 81; 45 and 82; 45 and 83;

45 and 84; 45 and 85; 45 and 86; 45 and 87; 46 and 65; 46 and 66; 46 and 67; 46 and 68; 46 and 69; 46 and 70; 46 and 71; 46 and 72; 46 and 73; 46 and 74; 46 and 75; 46 and 76; 46 and 77; 46 and 78; 46 and 79; 46 and 80; 46 and 81; 46 and 82; 46 and 83;

46 and 84; 46 and 85; 46 and 86; 46 and 87; 47 and 65; 47 and 66; 47 and 67; 47 and 68; 47 and 69; 47 and 70; 47 and 71; 47 and 72; 47 and 73; 47 and 74; 47 and 75; 47 and 76; 47 and 77; 47 and 78; 47 and 79; 47 and 80; 47 and 81; 47 and 82; 47 and 83;

47 and 84; 47 and 85; 47 and 86; 47 and 87; 48 and 65; 48 and 66; 48 and 67; 48 and 68; 48 and 69; 48 and 70; 48 and 71; 48 and 72; 48 and 73; 48 and 74; 48 and 75; 48 and 76; 48 and 77; 48 and 78; 48 and 79; 48 and 80; 48 and 81; 48 and 82; 48 and 83;

48 and 84; 48 and 85; 48 and 86; 48 and 87; 49 and 65; 49 and 66; 49 and 67; 49 and 68; 49 and 69; 49 and 70; 49 and 71; 49 and 72; 49 and 73; 49 and 74; 49 and 75; 49 and 76; 49 and 77; 49 and 78; 49 and 79; 49 and 80; 49 and 81; 49 and 82; 49 and 83;

49 and 84; 49 and 85; 49 and 86; 49 and 87; 50 and 65; 50 and 66; 50 and 67; 50 and 68; 50 and 69; 50 and 70; 50 and 71; 50 and 72; 50 and 73; 50 and 74; 50 and 75; 50 and 76; 50 and 77; 50 and 78; 50 and 79; 50 and 80; 50 and 81; 50 and 82; 50 and 83;

50 and 84; 50 and 85; 50 and 86; 50 and 87; 51 and 65; 51 and 66; 51 and 67; 51 and 68; 51 and 69; 51 and 70; 51 and 71; 51 and 72; 51 and 73; 51 and 74; 51 and 75; 51 and 76; 51 and 77; 51 and 78; 51 and 79; 51 and 80; 51 and 81; 51 and 82; 51 and 83;

51 and 84; 51 and 85; 51 and 86; 51 and 87; 52 and 65; 52 and 66; 52 and 67; 52 and 68; 52 and 69; 52 and 70; 52 and 71; 52 and 72; 52 and 73; 52 and 74; 52 and 75; 52 and 76; 52 and 77; 52 and 78; 52 and 79; 52 and 80; 52 and 81; 52 and 82; 52 and 83;

52 and 84; 52 and 85; 52 and 86; 52 and 87; 53 and 65; 53 and 66; 53 and 67; 53 and 68; 53 and 69; 53 and 70; 53 and 71; 53 and 72; 53 and 73; 53 and 74; 53 and 75; 53 and 76; 53 and 77; 53 and 78; 53 and 79; 53 and 80; 53 and 81; 53 and 82; 53 and 83;

53 and 84; 53 and 85; 53 and 86; 53 and 87; 54 and 65; 54 and 66; 54 and 67; 54 and 68; 54 and 69; 54 and 70; 54 and 71; 54 and 72; 54 and 73; 54 and 74; 54 and 75; 54 and 76; 54 and 77; 54 and 78; 54 and 79; 54 and 80; 54 and 81; 54 and 82; 54 and 83;

54 and 84; 54 and 85; 54 and 86; 54 and 87; 55 and 65; 55 and 66; 55 and 67; 55 and 68; 55 and 69; 55 and 70; 55 and 71; 55 and 72; 55 and 73; 55 and 74; 55 and 75; 55 and 76; 55 and 77; 55 and 78; 55 and 79; 55 and 80; 55 and 81; 55 and 82; 55 and 83;

55 and 84; 55 and 85; 55 and 86; 55 and 87; 56 and 65; 56 and 66; 56 and 67; 56 and 68; 56 and 69; 56 and 70; 56 and 71; 56 and 72; 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 56 and 78; 56 and 79; 56 and 80; 56 and 81; 56 and 82; 56 and 83;

56 and 84; 56 and 85; 56 and 86; 56 and 87; 57 and 65; 57 and 66; 57 and 67; 57 and 68; 57 and 69; 57 and 70; 57 and 71; 57 and 72; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 57 and 78; 57 and 79; 57 and 80; 57 and 81; 57 and 82; 57 and 83;

57 and 84; 57 and 85; 57 and 86; 57 and 87; 58 and 65; 58 and 66; 58 and 67; 58 and 68; 58 and 69; 58 and 70; 58 and 71; 58 and 72; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 58 and 78; 58 and 79; 58 and 80; 58 and 81; 58 and 82; 58 and 83;

58 and 84; 58 and 85; 58 and 86; 58 and 87; 59 and 65; 59 and 66; 59 and 67; 59 and 68; 59 and 69; 59 and 70; 59 and 71; 59 and 72; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 59 and 78; 59 and 79; 59 and 80; 59 and 81; 59 and 82; 59 and 83;

59 and 84; 59 and 85; 59 and 86; 59 and 87; 60 and 65; 60 and 66; 60 and 67; 60 and 68; 60 and 69; 60 and 70; 60 and 71; 60 and 72; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 60 and 78; 60 and 79; 60 and 80; 60 and 81; 60 and 82; 60 and 83;

60 and 84; 60 and 85; 60 and 86; 60 and 87; 61 and 65; 61 and 66; 61 and 67; 61 and 68; 61 and 69; 61 and 70; 61 and 71; 61 and 72; 61 and 73; 61 and 74; 61 and 75; 61 and 76; 61 and 77; 61 and 78; 61 and 79; 61 and 80; 61 and 81; 61 and 82; 61 and 83;

61 and 84; 61 and 85; 61 and 86; 61 and 87; 62 and 65; 62 and 66; 62 and 67; 62 and 68; 62 and 69; 62 and 70; 62 and 71; 62 and 72; 62 and 73; 62 and 74; 62 and 75; 62 and 76; 62 and 77; 62 and 78; 62 and 79; 62 and 80; 62 and 81; 62 and 82; 62 and 83;

62 and 84; 62 and 85; 62 and 86; 62 and 87; 63 and 65; 63 and 66; 63 and 67; 63 and 68; 63 and 69; 63 and 70; 63 and 71; 63 and 72; 63 and 73; 63 and 74; 63 and 75; 63 and 76; 63 and 77; 63 and 78; 63 and 79; 63 and 80; 63 and 81; 63 and 82; 63 and 83;

63 and 84; 63 and 85; 63 and 86; 63 and 87; 64 and 65; 64 and 66; 64 and 67; 64 and 68; 64 and 69; 64 and 70; 64 and 71; 64 and 72; 64 and 73; 64 and 74; 64 and 75; 64 and 76; 64 and 77; 64 and 78; 64 and 79; 64 and 80; 64 and 81; 64 and 82; 64 and 83;

64 and 84; 64 and 85; 64 and 86; or 64 and 87; b. SEQ ID NOs: 88 and 65; 88 and 66; 88 and 67; 88 and 68; 88 and 69; 88 and 70; 88 and 71; 88 and 72; 88 and 73; 88 and 74; 88 and 75; 88 and 76; 88 and 77; 88 and 78;

88 and 79; 88 and 80; 88 and 81; 88 and 82; 88 and 83; 88 and 84; 88 and 85; 88 and 86; 88 and 87; 89 and 65; 89 and 66; 89 and 67; 89 and 68; 89 and 69; 89 and 70; 89 and 71; 89 and 72; 89 and 73; 89 and 74; 89 and 75; 89 and 76; 89 and 77; 89 and 78;

89 and 79; 89 and 80; 89 and 81; 89 and 82; 89 and 83; 89 and 84; 89 and 85; 89 and 86; 89 and 87; 90 and 65; 90 and 66; 90 and 67; 90 and 68; 90 and 69; 90 and 70; 90 and 71; 90 and 72; 90 and 73; 90 and 74; 90 and 75; 90 and 76; 90 and 77; 90 and 78;

90 and 79; 90 and 80; 90 and 81; 90 and 82; 90 and 83; 90 and 84; 90 and 85; 90 and 86; 90 and 87; 91 and 65; 91 and 66; 91 and 67; 91 and 68; 91 and 69; 91 and 70; 91 and 71; 91 and 72; 91 and 73; 91 and 74; 91 and 75; 91 and 76; 91 and 77; 91 and 78;

91 and 79; 91 and 80; 91 and 81; 91 and 82; 91 and 83; 91 and 84; 91 and 85; 91 and 86; 91 and 87; 92 and 65; 92 and 66; 92 and 67; 92 and 68; 92 and 69; 92 and 70; 92 and 71; 92 and 72; 92 and 73; 92 and 74; 92 and 75; 92 and 76; 92 and 77; 92 and 78;

92 and 79; 92 and 80; 92 and 81; 92 and 82; 92 and 83; 92 and 84; 92 and 85; 92 and 86; 92 and 87; 93 and 65; 93 and 66; 93 and 67; 93 and 68; 93 and 69; 93 and 70; 93 and 71; 93 and 72; 93 and 73; 93 and 74; 93 and 75; 93 and 76; 93 and 77; 93 and 78;

93 and 79; 93 and 80; 93 and 81; 93 and 82; 93 and 83; 93 and 84; 93 and 85; 93 and 86; 93 and 87; 94 and 65; 94 and 66; 94 and 67; 94 and 68; 94 and 69; 94 and 70; 94 and 71; 94 and 72; 94 and 73; 94 and 74; 94 and 75; 94 and 76; 94 and 77; 94 and 78;

94 and 79; 94 and 80; 94 and 81; 94 and 82; 94 and 83; 94 and 84; 94 and 85; 94 and 86; 94 and 87; 95 and 65; 95 and 66; 95 and 67; 95 and 68; 95 and 69; 95 and 70; 95 and 71; 95 and 72; 95 and 73; 95 and 74; 95 and 75; 95 and 76; 95 and 77; 95 and 78; 95 and 79; 95 and 80; 95 and 81; 95 and 82; 95 and 83; 95 and 84; 95 and 85; 95 and 86; 95 and 87; 96 and 65; 96 and 66; 96 and 67; 96 and 68; 96 and 69; 96 and 70; 96 and 71; 96 and 72; 96 and 73; 96 and 74; 96 and 75; 96 and 76; 96 and 77; 96 and 78;

96 and 79; 96 and 80; 96 and 81; 96 and 82; 96 and 83; 96 and 84; 96 and 85; 96 and 86; 96 and 87; 97 and 65; 97 and 66; 97 and 67; 97 and 68; 97 and 69; 97 and 70; 97 and 71; 97 and 72; 97 and 73; 97 and 74; 97 and 75; 97 and 76; 97 and 77; 97 and 78;

97 and 79; 97 and 80; 97 and 81; 97 and 82; 97 and 83; 97 and 84; 97 and 85; 97 and 86; 97 and 87; 98 and 65; 98 and 66; 98 and 67; 98 and 68; 98 and 69; 98 and 70; 98 and 71; 98 and 72; 98 and 73; 98 and 74; 98 and 75; 98 and 76; 98 and 77; 98 and 78;

98 and 79; 98 and 80; 98 and 81; 98 and 82; 98 and 83; 98 and 84; 98 and 85; 98 and 86; 98 and 87; 99 and 65; 99 and 66; 99 and 67; 99 and 68; 99 and 69; 99 and 70; 99 and 71; 99 and 72; 99 and 73; 99 and 74; 99 and 75; 99 and 76; 99 and 77; 99 and 78;

99 and 79; 99 and 80; 99 and 81; 99 and 82; 99 and 83; 99 and 84; 99 and 85; 99 and 86; 99 and 87; 100 and 65; 100 and 66; 100 and 67; 100 and 68; 100 and 69; 100 and

70; 100 and 71; 100 and 72; 100 and 73; 100 and 74; 100 and 75; 100 and 76; 100 and 77; 100 and 78; 100 and 79; 100 and 80; 100 and 81; 100 and 82; 100 and 83;

100 and 84; 100 and 85; 100 and 86; 100 and 87; 101 and 65; 101 and 66; 101 and 67; 101 and 68; 101 and 69; 101 and 70; 101 and 71; 101 and 72; 101 and 73; 101 and 74; 101 and 75; 101 and 76; 101 and 77; 101 and 78; 101 and 79; 101 and 80;

101 and 81; 101 and 82; 101 and 83; 101 and 84; 101 and 85; 101 and 86; 101 and 87; 102 and 65; 102 and 66; 102 and 67; 102 and 68; 102 and 69; 102 and 70; 102 and 71; 102 and 72; 102 and 73; 102 and 74; 102 and 75; 102 and 76; 102 and 77;

102 and 78; 102 and 79; 102 and 80; 102 and 81; 102 and 82; 102 and 83; 102 and 84; 102 and 85; 102 and 86; 102 and 87; 103 and 65; 103 and 66; 103 and 67; 103 and 68; 103 and 69; 103 and 70; 103 and 71; 103 and 72; 103 and 73; 103 and 74;

103 and 75; 103 and 76; 103 and 77; 103 and 78; 103 and 79; 103 and 80; 103 and 81; 103 and 82; 103 and 83; 103 and 84; 103 and 85; 103 and 86; 103 and 87; 104 and 65; 104 and 66; 104 and 67; 104 and 68; 104 and 69; 104 and 70; 104 and 71;

104 and 72; 104 and 73; 104 and 74; 104 and 75; 104 and 76; 104 and 77; 104 and 78; 104 and 79; 104 and 80; 104 and 81; 104 and 82; 104 and 83; 104 and 84; 104 and 85; 104 and 86; 104 and 87; 105 and 65; 105 and 66; 105 and 67; 105 and 68;

105 and 69; 105 and 70; 105 and 71; 105 and 72; 105 and 73; 105 and 74; 105 and 75; 105 and 76; 105 and 77; 105 and 78; 105 and 79; 105 and 80; 105 and 81; 105 and 82; 105 and 83; 105 and 84; 105 and 85; 105 and 86; 105 and 87; 106 and 65;

106 and 66; 106 and 67; 106 and 68; 106 and 69; 106 and 70; 106 and 71; 106 and 72; 106 and 73; 106 and 74; 106 and 75; 106 and 76; 106 and 77; 106 and 78; 106 and 79; 106 and 80; 106 and 81; 106 and 82; 106 and 83; 106 and 84; 106 and 85; 106 and 86; 106 and 87; 107 and 65; 107 and 66; 107 and 67; 107 and 68; 107 and 69; 107 and 70; 107 and 71; 107 and 72; 107 and 73; 107 and 74; 107 and 75; 107 and 76; 107 and 77; 107 and 78; 107 and 79; 107 and 80; 107 and 81; 107 and 82;

107 and 83; 107 and 84; 107 and 85; 107 and 86; 107 and 87; 108 and 65; 108 and 66; 108 and 67; 108 and 68; 108 and 69; 108 and 70; 108 and 71; 108 and 72; 108 and 73; 108 and 74; 108 and 75; 108 and 76; 108 and 77; 108 and 78; 108 and 79;

108 and 80; 108 and 81; 108 and 82; 108 and 83; 108 and 84; 108 and 85; 108 and 86; 108 and 87; 109 and 65; 109 and 66; 109 and 67; 109 and 68; 109 and 69; 109 and 70; 109 and 71; 109 and 72; 109 and 73; 109 and 74; 109 and 75; 109 and 76;

109 and 77; 109 and 78; 109 and 79; 109 and 80; 109 and 81; 109 and 82; 109 and 83; 109 and 84; 109 and 85; 109 and 86; 109 and 87; 110 and 65; 110 and 66; 110 and 67; 110 and 68; 110 and 69; 110 and 70; 110 and 71; 110 and 72; 110 and 73;

110 and 74; 110 and 75; 110 and 76; 110 and 77; 110 and 78; 110 and 79; 110 and 80; 110 and 81; 110 and 82; 110 and 83; 110 and 84; 110 and 85; 110 and 86; 110 and 87; 111 and 65; 111 and 66; 111 and 67; 111 and 68; 111 and 69; 111 and 70;

111 and 71; 111 and 72; 111 and 73; 111 and 74; 111 and 75; 111 and 76; 111 and 77; 111 and 78; 111 and 79; 111 and 80; 111 and 81; 111 and 82; 111 and 83; 111 and 84; 111 and 85; 111 and 86; 111 and 87; 112 and 65; 112 and 66; 112 and 67;

112 and 68; 112 and 69; 112 and 70; 112 and 71; 112 and 72; 112 and 73; 112 and 74; 112 and 75; 112 and 76; 112 and 77; 112 and 78; 112 and 79; 112 and 80; 112 and 81; 112 and 82; 112 and 83; 112 and 84; 112 and 85; 112 and 86; 112 and 87;

113 and 65; 113 and 66; 113 and 67; 113 and 68; 113 and 69; 113 and 70; 113 and 71; 113 and 72; 113 and 73; 113 and 74; 113 and 75; 113 and 76; 113 and 77; 113 and 78; 113 and 79; 113 and 80; 113 and 81; 113 and 82; 113 and 83; 113 and 84;

113 and 85; 113 and 86; 113 and 87; 114 and 65; 114 and 66; 114 and 67; 114 and 68; 114 and 69; 114 and 70; 114 and 71; 114 and 72; 114 and 73; 114 and 74; 114 and 75; 114 and 76; 114 and 77; 114 and 78; 114 and 79; 114 and 80; 114 and 81;

114 and 82; 114 and 83; 114 and 84; 114 and 85; 114 and 86; 114 and 87; 115 and 65; 115 and 66; 115 and 67; 115 and 68; 115 and 69; 115 and 70; 115 and 71; 115 and 72; 115 and 73; 115 and 74; 115 and 75; 115 and 76; 115 and 77; 115 and 78;

115 and 79; 115 and 80; 115 and 81; 115 and 82; 115 and 83; 115 and 84; 115 and 85; 115 and 86; 115 and 87; 116 and 65; 116 and 66; 116 and 67; 116 and 68; 116 and 69; 116 and 70; 116 and 71; 116 and 72; 116 and 73; 116 and 74; 116 and 75;

116 and 76; 116 and 77; 116 and 78; 116 and 79; 116 and 80; 116 and 81; 116 and 82; 116 and 83; 116 and 84; 116 and 85; 116 and 86; 116 and 87; 117 and 65; 117 and 66; 117 and 67; 117 and 68; 117 and 69; 117 and 70; 117 and 71; 117 and 72;

117 and 73; 117 and 74; 117 and 75; 117 and 76; 117 and 77; 117 and 78; 117 and 79; 117 and 80; 117 and 81; 117 and 82; 117 and 83; 117 and 84; 117 and 85; 117 and 86; 117 and 87; 118 and 65; 118 and 66; 118 and 67; 118 and 68; 118 and 69;

118 and 70; 118 and 71; 118 and 72; 118 and 73; 118 and 74; 118 and 75; 118 and 76; 118 and 77; 118 and 78; 118 and 79; 118 and 80; 118 and 81; 118 and 82; 118 and 83; 118 and 84; 118 and 85; 118 and 86; 118 and 87; 119 and 65; 119 and 66;

119 and 67; 119 and 68; 119 and 69; 119 and 70; 119 and 71; 119 and 72; 119 and 73; 119 and 74; 119 and 75; 119 and 76; 119 and 77; 119 and 78; 119 and 79; 119 and 80; 119 and 81; 119 and 82; 119 and 83; 119 and 84; 119 and 85; 119 and 86;

119 and 87; 120 and 65; 120 and 66; 120 and 67; 120 and 68; 120 and 69; 120 and 70; 120 and 71; 120 and 72; 120 and 73; 120 and 74; 120 and 75; 120 and 76; 120 and 77; 120 and 78; 120 and 79; 120 and 80; 120 and 81; 120 and 82; 120 and 83;

120 and 84; 120 and 85; 120 and 86; 120 and 87; 121 and 65; 121 and 66; 121 and 67; 121 and 68; 121 and 69; 121 and 70; 121 and 71; 121 and 72; 121 and 73; 121 and 74; 121 and 75; 121 and 76; 121 and 77; 121 and 78; 121 and 79; 121 and 80;

121 and 81; 121 and 82; 121 and 83; 121 and 84; 121 and 85; 121 and 86; 121 and 87; 122 and 65; 122 and 66; 122 and 67; 122 and 68; 122 and 69; 122 and 70; 122 and 71; 122 and 72; 122 and 73; 122 and 74; 122 and 75; 122 and 76; 122 and 77;

122 and 78; 122 and 79; 122 and 80; 122 and 81; 122 and 82; 122 and 83; 122 and 84; 122 and 85; 122 and 86; 122 and 87; 123 and 65; 123 and 66; 123 and 67; 123 and 68; 123 and 69; 123 and 70; 123 and 71; 123 and 72; 123 and 73; 123 and 74;

123 and 75; 123 and 76; 123 and 77; 123 and 78; 123 and 79; 123 and 80; 123 and 81; 123 and 82; 123 and 83; 123 and 84; 123 and 85; 123 and 86; 123 and 87; 124 and 65; 124 and 66; 124 and 67; 124 and 68; 124 and 69; 124 and 70; 124 and 71;

124 and 72; 124 and 73; 124 and 74; 124 and 75; 124 and 76; 124 and 77; 124 and 78; 124 and 79; 124 and 80; 124 and 81; 124 and 82; 124 and 83; 124 and 84; 124 and 85; 124 and 86; 124 and 87; 125 and 65; 125 and 66; 125 and 67; 125 and 68;

125 and 69; 125 and 70; 125 and 71; 125 and 72; 125 and 73; 125 and 74; 125 and 75; 125 and 76; 125 and 77; 125 and 78; 125 and 79; 125 and 80; 125 and 81; 125 and 82; 125 and 83; 125 and 84; 125 and 85; 125 and 86; 125 and 87; 126 and 65;

126 and 66; 126 and 67; 126 and 68; 126 and 69; 126 and 70; 126 and 71; 126 and 72; 126 and 73; 126 and 74; 126 and 75; 126 and 76; 126 and 77; 126 and 78; 126 and 79; 126 and 80; 126 and 81; 126 and 82; 126 and 83; 126 and 84; 126 and 85;

126 and 86; 126 and 87; 127 and 65; 127 and 66; 127 and 67; 127 and 68; 127 and 69; 127 and 70; 127 and 71; 127 and 72; 127 and 73; 127 and 74; 127 and 75; 127 and 76; 127 and 77; 127 and 78; 127 and 79; 127 and 80; 127 and 81; 127 and 82;

127 and 83; 127 and 84; 127 and 85; 127 and 86; 127 and 87; 128 and 65; 128 and 66; 128 and 67; 128 and 68; 128 and 69; 128 and 70; 128 and 71; 128 and 72; 128 and 73; 128 and 74; 128 and 75; 128 and 76; 128 and 77; 128 and 78; 128 and 79;

128 and 80; 128 and 81; 128 and 82; 128 and 83; 128 and 84; 128 and 85; 128 and 86; 128 and 87; 129 and 65; 129 and 66; 129 and 67; 129 and 68; 129 and 69; 129 and 70; 129 and 71; 129 and 72; 129 and 73; 129 and 74; 129 and 75; 129 and 76;

129 and 77; 129 and 78; 129 and 79; 129 and 80; 129 and 81; 129 and 82; 129 and 83; 129 and 84; 129 and 85; 129 and 86; 129 and 87; 130 and 65; 130 and 66; 130 and 67; 130 and 68; 130 and 69; 130 and 70; 130 and 71; 130 and 72; 130 and 73;

130 and 74; 130 and 75; 130 and 76; 130 and 77; 130 and 78; 130 and 79; 130 and 80; 130 and 81; 130 and 82; 130 and 83; 130 and 84; 130 and 85; 130 and 86; 130 and 87; 131 and 65; 131 and 66; 131 and 67; 131 and 68; 131 and 69; 131 and 70;

131 and 71; 131 and 72; 131 and 73; 131 and 74; 131 and 75; 131 and 76; 131 and 77; 131 and 78; 131 and 79; 131 and 80; 131 and 81; 131 and 82; 131 and 83; 131 and 84; 131 and 85; 131 and 86; 131 and 87; 132 and 65; 132 and 66; 132 and 67;

132 and 68; 132 and 69; 132 and 70; 132 and 71; 132 and 72; 132 and 73; 132 and 74; 132 and 75; 132 and 76; 132 and 77; 132 and 78; 132 and 79; 132 and 80; 132 and 81; 132 and 82; 132 and 83; 132 and 84; 132 and 85; 132 and 86; 132 and 87;

133 and 65; 133 and 66; 133 and 67; 133 and 68; 133 and 69; 133 and 70; 133 and 71; 133 and 72; 133 and 73; 133 and 74; 133 and 75; 133 and 76; 133 and 77; 133 and 78; 133 and 79; 133 and 80; 133 and 81; 133 and 82; 133 and 83; 133 and 84;

133 and 85; 133 and 86; 133 and 87; 134 and 65; 134 and 66; 134 and 67; 134 and 68; 134 and 69; 134 and 70; 134 and 71; 134 and 72; 134 and 73; 134 and 74; 134 and 75; 134 and 76; 134 and 77; 134 and 78; 134 and 79; 134 and 80; 134 and 81;

134 and 82; 134 and 83; 134 and 84; 134 and 85; 134 and 86; 134 and 87; 135 and 65; 135 and 66; 135 and 67; 135 and 68; 135 and 69; 135 and 70; 135 and 71; 135 and 72; 135 and 73; 135 and 74; 135 and 75; 135 and 76; 135 and 77; 135 and 78;

135 and 79; 135 and 80; 135 and 81; 135 and 82; 135 and 83; 135 and 84; 135 and 85; 135 and 86; 135 and 87; 136 and 65; 136 and 66; 136 and 67; 136 and 68; 136 and 69; 136 and 70; 136 and 71; 136 and 72; 136 and 73; 136 and 74; 136 and 75;

136 and 76; 136 and 77; 136 and 78; 136 and 79; 136 and 80; 136 and 81; 136 and 82; 136 and 83; 136 and 84; 136 and 85; 136 and 86; 136 and 87; 137 and 65; 137 and 66; 137 and 67; 137 and 68; 137 and 69; 137 and 70; 137 and 71; 137 and 72;

137 and 73; 137 and 74; 137 and 75; 137 and 76; 137 and 77; 137 and 78; 137 and 79; 137 and 80; 137 and 81; 137 and 82; 137 and 83; 137 and 84; 137 and 85; 137 and 86; 137 and 87; 138 and 65; 138 and 66; 138 and 67; 138 and 68; 138 and 69;

138 and 70; 138 and 71; 138 and 72; 138 and 73; 138 and 74; 138 and 75; 138 and 76; 138 and 77; 138 and 78; 138 and 79; 138 and 80; 138 and 81; 138 and 82; 138 and 83; 138 and 84; 138 and 85; 138 and 86; 138 and 87; 139 and 65; 139 and 66; 139 and 67; 139 and 68; 139 and 69; 139 and 70; 139 and 71; 139 and 72; 139 and 73; 139 and 74; 139 and 75; 139 and 76; 139 and 77; 139 and 78; 139 and 79; 139 and 80; 139 and 81; 139 and 82; 139 and 83; 139 and 84; 139 and 85; 139 and 86;

139 and 87; 140 and 65; 140 and 66; 140 and 67; 140 and 68; 140 and 69; 140 and 70; 140 and 71; 140 and 72; 140 and 73; 140 and 74; 140 and 75; 140 and 76; 140 and 77; 140 and 78; 140 and 79; 140 and 80; 140 and 81; 140 and 82; 140 and 83;

140 and 84; 140 and 85; 140 and 86; 140 and 87; 141 and 65; 141 and 66; 141 and 67; 141 and 68; 141 and 69; 141 and 70; 141 and 71; 141 and 72; 141 and 73; 141 and 74; 141 and 75; 141 and 76; 141 and 77; 141 and 78; 141 and 79; 141 and 80;

141 and 81; 141 and 82; 141 and 83; 141 and 84; 141 and 85; 141 and 86; 141 and 87; 142 and 65; 142 and 66; 142 and 67; 142 and 68; 142 and 69; 142 and 70; 142 and 71; 142 and 72; 142 and 73; 142 and 74; 142 and 75; 142 and 76; 142 and 77;

142 and 78; 142 and 79; 142 and 80; 142 and 81; 142 and 82; 142 and 83; 142 and 84; 142 and 85; 142 and 86; 142 and 87; 143 and 65; 143 and 66; 143 and 67; 143 and 68; 143 and 69; 143 and 70; 143 and 71; 143 and 72; 143 and 73; 143 and 74;

143 and 75; 143 and 76; 143 and 77; 143 and 78; 143 and 79; 143 and 80; 143 and 81; 143 and 82; 143 and 83; 143 and 84; 143 and 85; 143 and 86; or 143 and 87; c. SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and 80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70;

57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76;

58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; or d. SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82; 121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and 83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and

84; 119 and 84; 120 and 84; 121 and 84; 90 and 84; 115 and 85; 116 and 85; 117 and

85; 118 and 85; 119 and 85; 120 and 85; 121 and 85; 90 and 85; 115 and 68; 116 and

68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and 68; 90 and 68; 115 and

69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and 70; 90 and 70; 115 and 71; 116 and 71; 117 and 71; 118 and 71; 119 and 71; 120 and 71; 121 and 71; 90 and 71; 115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and 75; 116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76; 116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and 81; 117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81.

20. A composition comprising one or more nucleic acid molecules encoding a. a Staphylococcus lugdunensis Cas9 (SluCas9); and b. at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first guide sequence and a second guide sequence selected from any of the pairs of guide sequences shown in Tables IE or 1F.

21. A composition comprising: a. a single nucleic acid molecule comprising: i. a nucleic acid encoding Staphylococcus lugdunensis (SluCas9) and at least one, at least two, or at least three guide RNAs; or ii. a nucleic acid encoding SluCas9 and from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or iii. a nucleic acid encoding SluCas9 and 1, 2, or 3 guide RNAs; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A-1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide sequence selected from the pairs of guide sequences shown in Tables 1E or 1F, and further optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence; or b. two nucleic acid molecules comprising: i. a first nucleic acid encoding SluCas9; and a second nucleic acid that does not encode a SluCas9 and encodes any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; ii. a first nucleic acid encoding SluCas9 and

1. at least one, at least two, or at least three guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. 1, 2, or 3 guide RNAs; and a second nucleic acid that does not encode a SluCas9, optionally wherein the second nucleic acid comprises any one of the following:

1. at least one, at least two, at least three, at least four, at least five, or at least six guide RNAs; or

2. from one to n guide RNAs, wherein n is no more than the maximum number of guide RNAs that can be expressed from said nucleic acid; or

3. from one to six guide RNAs; iii. a first nucleic acid encoding SluCas9 and at least one, at least two, or at least three guide RNAs; and a second nucleic acid that does not encode a SluCas9 and encodes from one to six guide RNAs; or iv. a first nucleic acid encoding SluCas9 and at least two guide RNAs, and a second nucleic acid that does not encode a SluCas9 and encodes at least one additional copy of each of the guide RNAs encoded in the first nucleic acid; wherein each guide RNA comprises a guide sequence selected from any one of the guide sequences of Tables 1A-1C, optionally wherein the composition comprises at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a guide sequence selected from the pairs of guide sequences shown in Tables 1E or 1F, and optionally wherein a first guide RNA of the at least one pair of guide RNAs binds upstream of a target sequence and a second guide RNA of the at least one pair of guide RNAs binds downstream of the target sequence.

22. A composition comprising a single nucleic acid molecule encoding at least one pair of guide RNAs and a Cas9, wherein the single nucleic acid molecule comprises: a. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences shown in Table 1E or 1F; and a second nucleic acid encoding a Staphylococcus lugdunensis Cas9 (SluCas9); b. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences of: SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and 80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70; 57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76; 58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; and a second nucleic acid encoding a SluCas9; or c. a first nucleic acid encoding the at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a pair of guide sequences selected from any one of the pairs of guide sequences of: SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82; 121 and 82; 90 and 82; 115 and 83; 116 and 83;

117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and 83; 90 and 83; 115 and 84;

116 and 84; 117 and 84; 118 and 84; 119 and 84; 120 and 84; 121 and 84; 90 and 84;

115 and 85; 116 and 85; 117 and 85; 118 and 85; 119 and 85; 120 and 85; 121 and

85; 90 and 85; 115 and 68; 116 and 68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and 68; 90 and 68; 115 and 69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and

70; 119 and 70; 120 and 70; 121 and 70; 90 and 70; 115 and 71; 116 and 71; 117 and

71; 118 and 71; 119 and 71; 120 and 71; 121 and 71; 90 and 71; 115 and 73; 116 and

73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and

74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and 75; 116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76; 116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and 81; 117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81; and a second nucleic acid encoding a SluCas9.

23. A composition comprising a first and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a Staphylococcus lugdunensis Cas9 (SluCas9) and optionally a first or a first and second guide RNA, and the second nucleic acid molecule comprises a first or a first and second guide RNA, wherein the first guide RNA comprises a first guide sequence and the second guide RNA comprises a second guide sequence selected from any one the following pairs of first and second guide sequences: a. any one of the pairs of first and second guide sequences shown in Tables IE or 1F; b. SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and 80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70;

57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76;

58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; or c. SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and 82; 121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and 83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and

84; 119 and 84; 120 and 84; 121 and 84; 90 and 84; 115 and 85; 116 and 85; 117 and

85; 118 and 85; 119 and 85; 120 and 85; 121 and 85; 90 and 85; 115 and 68; 116 and

68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and 68; 90 and 68; 115 and

69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and 70; 90 and 70; 115 and 71; 116 and 71; 117 and 71; 118 and 71; 119 and 71; 120 and 71; 121 and 71; 90 and 71; 115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and 75; 116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76; 116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and 81; 117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81.

24. A composition comprising one or more nucleic acid molecules encoding a Staphylococcus lugdunensis Cas9 (SluCas9) and at least one pair of guide RNAs, wherein the at least one pair of guide RNAs comprises a first and a second guide RNA, wherein the first guide RNA targets a sequence in intron 53 of a DMD gene and the second guide RNA targets a sequence in exon 53 of the DMD gene, or the first guide RNA targets a sequence in exon 53 of a DMD gene and the second guide RNA targets a sequence in intron 52 of the DMD gene.

25. The composition of any one of claims 12-24, comprising at least one pair of guide RNAs, wherein the at least one pair of guide RNAs is capable of excising a fragment of the DMD gene, wherein the fragment comprises a splice donor site or a splice acceptor site, and optionally wherein the fragment is between 5 and 400 nucleotides in length.

26. The composition of any one of claims 12-15 or 25, wherein the excised fragment of the DMD gene does not comprise an entire exon of the DMD gene.

27. The composition of any one of claims 12-26, comprising at least one pair of guide RNAs, wherein the first and the second guide RNAs of the at least one pair of guide RNAs each comprise a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a first and a second guide sequence selected from any one of the following pairs of first and second guide sequences: a. any one of the pairs of first and second guide sequences shown in Tables IE or 1F; b. SEQ ID NOs: 2 and 85; 2 and 71; 2 and 84; 2 and 70; 2 and 83; 2 and 82; 2 and 81; 2 and 80; 2 and 69; 2 and 68; 2 and 76; 2 and 75; 2 and 74; 2 and 73; 35 and 85; 35 and 71; 35 and 84; 36 and 85; 36 and 71; 36 and 84; 35 and 70; 37 and 85; 37 and 71; 37 and 84; 36 and 70; 37 and 70; 38 and 85; 38 and 71; 39 and 85; 39 and 71; 38 and 84; 39 and 84; 35 and 83; 38 and 70; 39 and 70; 36 and 83; 37 and 83; 35 and 82; 35 and 81; 36 and 82; 38 and 83; 39 and 83; 36 and 81; 37 and 82; 35 and 80; 37 and 81; 36 and 80; 38 and 82; 39 and 82; 37 and 80; 38 and 81; 39 and 81; 38 and 80; 39 and 80; 35 and 69; 35 and 68; 36 and 69; 35 and 76; 37 and 69; 36 and 68; 35 and 75; 36 and 76; 35 and 74; 35 and 73; 37 and 68; 36 and 75; 37 and 76; 36 and 74; 38 and 69; 36 and 73; 39 and 69; 37 and 75; 37 and 74; 37 and 73; 38 and 68; 39 and 68; 38 and 76; 39 and 76; 38 and 75; 39 and 75; 38 and 74; 38 and 73; 39 and 74; 39 and 73; 57 and 85; 57 and 71; 58 and 85; 19 and 85; 58 and 71; 19 and 71; 57 and 84; 58 and 84; 19 and 84; 57 and 70; 58 and 70; 19 and 70; 20 and 85; 20 and 71; 20 and 84; 20 and 70;

57 and 83; 58 and 83; 19 and 83; 57 and 82; 58 and 82; 19 and 82; 20 and 83; 57 and 81; 58 and 81; 19 and 81; 57 and 80; 58 and 80; 19 and 80; 20 and 82; 20 and 81; 20 and 80; 57 and 69; 58 and 69; 19 and 69; 57 and 68; 58 and 68; 19 and 68; 57 and 76;

58 and 76; 19 and 76; 57 and 75; 58 and 75; 19 and 75; 20 and 69; 57 and 74; 57 and 73; 58 and 74; 19 and 74; 58 and 73; 19 and 73; 20 and 68; 20 and 76; 20 and 75; 20 and 74; or 20 and 73; or c. SEQ ID NOs: 115 and 82; 116 and 82; 117 and 82; 118 and 82; 119 and 82; 120 and

82; 121 and 82; 90 and 82; 115 and 83; 116 and 83; 117 and 83; 118 and 83; 119 and 83; 120 and 83; 121 and 83; 90 and 83; 115 and 84; 116 and 84; 117 and 84; 118 and

84; 119 and 84; 120 and 84; 121 and 84; 90 and 84; 115 and 85; 116 and 85; 117 and

85; 118 and 85; 119 and 85; 120 and 85; 121 and 85; 90 and 85; 115 and 68; 116 and

68; 117 and 68; 118 and 68; 119 and 68; 120 and 68; 121 and 68; 90 and 68; 115 and

69; 116 and 69; 117 and 69; 118 and 69; 119 and 69; 120 and 69; 121 and 69; 90 and 69; 115 and 70; 116 and 70; 117 and 70; 118 and 70; 119 and 70; 120 and 70; 121 and 70; 90 and 70; 115 and 71; 116 and 71; 117 and 71; 118 and 71; 119 and 71; 120 and 71; 121 and 71; 90 and 71; 115 and 73; 116 and 73; 117 and 73; 118 and 73; 119 and 73; 120 and 73; 121 and 73; 90 and 73; 115 and 74; 116 and 74; 117 and 74; 118 and 74; 119 and 74; 120 and 74; 121 and 74; 90 and 74; 115 and 75; 116 and 75; 117 and 75; 118 and 75; 119 and 75; 120 and 75; 121 and 75; 90 and 75; 115 and 76; 116 and 76; 117 and 76; 118 and 76; 119 and 76; 120 and 76; 121 and 76; 90 and 76; 115 and 80; 116 and 80; 117 and 80; 118 and 80; 119 and 80; 120 and 80; 121 and 80; 90 and 80; 115 and 81; 116 and 81; 117 and 81; 118 and 81; 119 and 81; 120 and 81; 121 and 81; or 90 and 81.

28. The composition of any one of claims 12-26, wherein: i) the first guide RNA comprises the sequence of SEQ ID NO: 70 and the second guide RNA comprises the sequence of SEQ ID NO: 2; ii) the first guide RNA comprises the sequence of SEQ ID NO: 76, and the second guide RNA comprises the sequence of SEQ ID NO: 2; or iii) the first guide RNA comprises the sequence of SEQ ID NO: 115, and the second guide RNA comprises the sequence of SEQ ID NO: 70.

29. The composition of any one of the preceding claims, comprising at least one pair of guide RNAs, wherein once expressed in vitro or in vivo in combination with a SluCas9 endonuclease, the at least one pair of guide RNAs (i) excises exon 53 of a DMD gene, (ii) excises a fragment of the DMD gene comprising a portion of intron 53 and an adjoining portion of exon 53, wherein the excised fragment of the DMD gene comprises a splice donor site, or (iii) excises a fragment of the DMD gene comprising a portion of exon 53 and an adjoining portion of intron 52, wherein the excised fragment of the DMD gene comprises a splice acceptor site; wherein the size of the excised fragment of the DMD gene is between 5 and 400 nucleotides in length.

30. The composition of any one of claims 12-15 or 25-29, wherein the size of the excised fragment of the DMD gene is between 5 and 400, 5 and 375, 5 and 350, 5 and 325, 5 and 300, 5 and 275, 5 and 250, 5 and 225, 5 and 200, 5 and 175, 5 and 150, 5 and 125, 5 and 100, 20 and 400, 20 and 375, 20 and 350, 20 and 325, 20 and 300, 20 and 275, 20 and 250, 20 and 225, 20 and 200, 20 and 175, 20 and 150, 20 and 125, 20 and 100, 50 and 400, 50 and 375, 50 and 350, 50 and 325, 50 and 300, 50 and 275, 50 and 250, 50 and 225, 50 and 200, 50 and 175, 50 and 150, 50 and 125, or 50 and 100 nucleotides in length.

31. The composition of any one of the preceding claims, wherein the one or more guide RNAs is an sgRNA, the first and/or second guide RNA is an sgRNA, or the at least one pair of guide RNAs is a pair of sgRNAs.

32. The composition of any one of the preceding claims, wherein the one or more guide RNAs is modified, the first and/or second guide RNA is modified, or the at least one pair of guide RNAs is a pair of modified guide RNAs.

33. The composition of any one of the preceding claims, wherein the one or more guide RNAs, the first and/or second guide RNA, the at least one pair of guide RNAs, or the nucleic acid is in a vector.

34. The composition of claim 33, wherein the vector is a lipid nanoparticle or a liposome.

35. The composition of claim 33, wherein the vector is a viral vector.

36. The composition of claim 35, wherein the viral vector is an AAV vector.

37. The composition of claim 36, wherein the AAV vector is an AAV9 vector.

38. The composition of any one of the preceding claims, wherein a promoter for the one or more guide RNAs, the first and/or second guide RNA, or the at least one pair of guide RNAs is hU6c.

39. The composition of any one of the preceding claims, wherein the one or more guide RNAs is a guide RNA for SluCas9, the first and/or second guide RNA is a guide RNA for SluCas9, or the at least one pair of guide RNAs is a pair of guide RNAs for SluCas9; and the one or more guide RNAs, the first and/or second guide RNA, or the at least one pair of guide RNAs comprise a scaffold comprising the sequence of any one of SEQ ID NOs: 600, 601, or 900- 917.

40. The composition of the immediately preceding claim, wherein the scaffold comprises the sequence of SEQ ID NO: 901.

41. The composition of any one of the preceding claims, wherein the one or more guide RNAs, the first and/or second guide RNA, or the at least one pair of guide RNAs is in an AAV vector, wherein the vector comprises from 5’ to 3’ with respect to the plus strand: the reverse complement of a first guide RNA scaffold sequence; the reverse complement of a nucleic acid encoding a first guide RNA sequence; the reverse complement of a promoter for expression of the nucleic acid encoding the first guide RNA sequence; a promoter for expression of a nucleic acid encoding SluCas9; a nucleic acid encoding a SluCas9; a polyadenylation sequence; a promoter for expression of a second guide RNA sequence in the same direction as the promoter for SluCas9; a second RNA guide sequence; and a second guide RNA scaffold sequence.

42. The composition of any one of the preceding claims, wherein the one or more guide RNAs, or the first and/or second guide RNA, is no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, or no more than 22 nucleotides in length.

43. A method of treating Duchenne Muscular Dystrophy (DMD), the method comprising delivering to a cell the composition of any one of claims 1-42.

44. A method of excising a DMD gene or a fragment of the DMD gene, the method comprising delivering to a cell the composition of any one of claims 1-42, wherein the size of the excised portion of the DMD gene is less than about 400 nucleotides.

45. A composition comprising: a) a pair of guide RNA molecules or a nucleic acid encoding a pair of RNA molecules, wherein the first guide RNA targets a region upstream of a splice acceptor site or a splice donor site in a DMD gene and wherein the second guide RNA targets a region downstream of a splice acceptor site or a splice donor site in the DMD gene; and b) an endonuclease or a nucleic acid encoding an endonuclease, optionally wherein the endonuclease is a Staphylococcus lugdunensis Cas9 (SluCas9).

46. The composition of claim 45, wherein the first guide RNA is in an intron of the DMD gene and the second guide RNA is in an exon of the DMD gene.

47. The composition of claim 45, wherein the first guide RNA is in an exon of the DMD gene and the second guide RNA is in an intron of the DMD gene.

48. The composition of claim 45, wherein the first guide RNA is in an exon of the DMD gene and the second guide RNA is in an exon of the DMD gene.

49. The composition of claim 45, wherein the first guide RNA is in an intron of the DMD gene and the second guide RNA is in an intron of the DMD gene.

50. The composition of any one of claims 45-49, wherein the splice acceptor site or splice donor site is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

74, 75, 76, 77, 78, or 78 of the DMD gene.

51. The composition of any one of claims 45-50, wherein the splice acceptor site or splice donor site is in intron 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57 of the DMD gene.

52. The composition of any one of claims 45-51, wherein the first guide RNA targets a region within 1-200, 1-150, 1-100, 1-50, 30-200, 30-150, 30-100, 30-50, 75-200, 75-150, 75-100, 100-200, 100-150, or 150-200 nucleotides upstream of the splice acceptor or splice donor site.

53. The composition of any one of claims 45-52, wherein the first guide RNA targets a region within 1-200, 1-150, 1-100, 1-50, 30-200, 30-150, 30-100, 30-50, 75-200, 75-150, 75-100, 100-200, 100-150, or 150-200 nucleotides downstream of the splice acceptor or splice donor site.

54. A method of excising a fragment of a DMD gene, the method comprising delivering to a cell the composition of any one of claims 45-53, wherein the pair of guide RNAs and the endonuclease excise a fragment of the DMD gene comprising the splice acceptor site or the splice donor site, wherein the fragment is between 3 and 400 nucleotides in length.

55. The method of claim 54, wherein the excised fragment of the DMD gene comprises a splice acceptor site.

56. The method of claim 54, wherein the excised fragment of the DMD gene comprises a splice donor site.

57. The method of any one of claims 54-56, wherein the excised fragment is between 3-200, 3- 150, 3-100, 3-50, 30-250, 30-200, 30-150, 30-100, 30-50, 75-250, 75-200, 75-150, 75-100, 100-250, 100-200, 100-150, 150-250, 150-200, 200-250, 250-400, 300-400, or 350-400 nucleotides in length.