TW202444388A - Modified guide rnas - Google Patents

Abstract

The present disclosure relates to guide RNAs that have been modified to improve performance and stability. In certain embodiments, a guide RNA of the present disclosure includes one or more modifications at its 5’ terminus and/or one or more modifications at its 3’ terminus.

Description

Translated fromChinese

經修飾的引導　RNAModified guide RNA

本揭露涉及已被修飾以改善穩定性及改善性能的引導 RNA。The present disclosure relates to guide RNAs that have been modified to improve stability and to improve performance.

規律間隔重複短迴文序列簇 (CRISPR)/CRISPR 相關蛋白 (Cas) 系統被廣泛用於編輯各種細胞類型之基因體。CRISPR/Cas 系統可分為兩類 (I 類及 II 類)，該兩類又可進一步細分為至少六種不同的類型：I 型、II 型、III 型、IV 型、V 型及 VI 型。在 II 型 CRISPR/Cas 系統中，Cas 蛋白 (諸如 Cas9) 與引導 RNA (gRNA) 分子形成複合物，且與具有原間隔序列相鄰基序 (PAM) 及間隔子之標靶核酸結合。gRNA 分子包括與標靶核酸中之間隔子互補且起到將 Cas 蛋白引導至標靶核酸之作用的序列。Cas 蛋白:gRNA 複合物對標靶核苷酸之識別及結合誘導標靶核酸發生裂解。Clustered regularly interspaced repeats (CRISPR)/CRISPR-associated protein (Cas) systems are widely used to edit the genome of various cell types. CRISPR/Cas systems can be divided into two categories (type I and type II), which can be further divided into at least six different types: type I, type II, type III, type IV, type V, and type VI. In type II CRISPR/Cas systems, Cas proteins (such as Cas9) form a complex with guide RNA (gRNA) molecules and bind to a target nucleic acid having a protospacer adjacent motif (PAM) and a spacer. The gRNA molecule includes a sequence that complements the spacer in the target nucleic acid and serves to guide the Cas protein to the target nucleic acid. The Cas protein:gRNA complex recognizes the target nucleotide and binds to induce the cleavage of the target nucleic acid.

已發現 gRNA 分子可在細胞中藉由核酸酶裂解 (例如，核酸內切酶或核酸外切酶裂解) 發生降解，此可能會影響 CRISPR-Cas 系統之功效。因此，此項技術中需要具有改善的穩定性及改善的基因編輯效率之 gRNA 分子。It has been found that gRNA molecules can be degraded in cells by nuclease cleavage(e.g., endonuclease or exonuclease cleavage), which may affect the efficacy of the CRISPR-Cas system. Therefore, gRNA molecules with improved stability and improved gene editing efficiency are needed in this technology.

本揭露內容提供經修飾之引導 RNA 分子。在某些實施例中，該引導 RNA 分子包括：(i) 在該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結，(ii) 在該引導 RNA 分子的 3' 端 (第「n」個) 核苷酸與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結，或 (iii) 在該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結以及在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。The present disclosure provides modified guide RNA molecules. In certain embodiments, the guide RNA molecule comprises: (i) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule, (ii) a phosphorodithioate bond between the 3' ("n"th) nucleotide and the n-1th nucleotide of the guide RNA molecule, or (iii) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule and a phosphorodithioate bond between the nth and n-1th nucleotides of the guide RNA molecule.

在某些實施例中，該引導 RNA 分子進一步包括在該引導 RNA 分子的 5' 端處的第二個與第三個核苷酸以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，該引導 RNA 分子包括在該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，該引導 RNA 分子包括在該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、第三個與第四個核苷酸、以及第四個與第五個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the guide RNA molecule further comprises a phosphorodithioate bond between the second and third nucleotides and the third and fourth nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises a phosphorodithioate bond between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises a phosphorodithioate bond between the first and second nucleotides, the second and third nucleotides, the third and fourth nucleotides, and the fourth and fifth nucleotides at the 5' end of the guide RNA molecule.

在某些實施例中，該引導 RNA 分子包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸或者第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，該引導 RNA 分子包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸、第 n-1 個與第 n-2 個核苷酸、以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，該引導 RNA 分子包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸、第 n-1 個與第 n-2 個核苷酸、第 n-2 個與第 n-3 個核苷酸、以及第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the guide RNA molecule comprises a phosphorothioate bond between the nth and n-1th nucleotides and the n-1th and n-2th nucleotides or the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises a phosphorothioate bond between the nth and n-1th nucleotides, the n-1th and n-2th nucleotides, and the n-2th and n-3th nucleotides of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises phosphorodithioate bonds between the n-th and n-1-th nucleotides, the n-1-th and n-2-th nucleotides, the n-2-th and n-3-th nucleotides, and the n-3-th and n-4-th nucleotides of the guide RNA molecule.

在某些實施例中，在該引導 RNA 分子的 5' 端處的第一個核苷酸包括選自由以下所組成之群組的修飾：(a) 經 2'-氟修飾的核苷酸、(b) 經 2'-O-甲基修飾的核苷酸、(c) 經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、(d) 鎖核酸 (LNA)、(e) 去氧核糖核苷酸，以及 (a) 至 (e) 中之兩者或更多者之組合。在某些實施例中，在該引導 RNA 分子的 5' 端處的第一個核苷酸包含經 2'-氟修飾的核苷酸。在某些實施例中，在該引導 RNA 分子的 5' 端處的第一個核苷酸包含經 2'-O-甲基修飾的核苷酸。在某些實施例中，在該引導 RNA 分子的 5' 端處的第一個核苷酸包含經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，在該引導 RNA 分子的 5' 端處的第一個核苷酸包含 LNA。在某些實施例中，在該引導 RNA 分子的 5' 端處的第一個核苷酸包含去氧核糖核苷酸。In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule comprises a modification selected from the group consisting of: (a) a 2'-fluoro modified nucleotide, (b) a 2'-O-methyl modified nucleotide, (c) a 2'-O-(2-methoxyethyl) modified nucleotide, (d) a locked nucleic acid (LNA), (e) a deoxyribonucleotide, and a combination of two or more of (a) to (e). In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-fluoro modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-O-methyl modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl) modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule comprises an LNA. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule comprises a deoxyribonucleotide.

在某些實施例中，該引導 RNA 分子的 5' 端處的 (i) 第二個核苷酸、(ii) 第三個核苷酸、(iii) 第四個核苷酸、(iv) 第二個與第三個核苷酸、(v) 第二個與第四個核苷酸、(vi) 第三個與第四個核苷酸或 (v) 第二個、第三個與第四個核苷酸包括選自由以下所組成之群組的修飾：(a) 經 2'-氟修飾的核苷酸、(b) 經 2'-O-甲基修飾的核苷酸、(c) 經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、(d) 鎖核酸 (LNA)、(e) 去氧核糖核苷酸，以及 (a) 至 (e) 中之兩者或更多者之組合。In certain embodiments, (i) the second nucleotide, (ii) the third nucleotide, (iii) the fourth nucleotide, (iv) the second and third nucleotides, (v) the second and fourth nucleotides, (vi) the third and fourth nucleotides, or (v) the second, third, and fourth nucleotides at the 5' end of the guide RNA molecule include a modification selected from the group consisting of: (a) 2'-fluoro-modified nucleotides, (b) 2'-O-methyl-modified nucleotides, (c) 2'-O-(2-methoxyethyl)-modified nucleotides, (d) locked nucleic acids (LNA), (e) deoxyribonucleotides, and a combination of two or more of (a) to (e).

在某些實施例中，該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包括三個連續的經 2'-氟修飾的核苷酸。在某些實施例中，該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包括三個連續的經 2'-O-甲基修飾的核苷酸。在某些實施例中，該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前四個核苷酸處包括四個連續的經 2'-O-甲基修飾的核苷酸。在某些實施例中，該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包括三個連續的經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包括三個連續的 LNA。在某些實施例中，該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包括三個連續的去氧核糖核苷酸。In certain embodiments, the guide RNA molecule comprises three consecutive 2'-fluorine-modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises three consecutive 2'-O-methyl-modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises four consecutive 2'-O-methyl-modified nucleotides at the first four nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises three consecutive 2'-O-(2-methoxyethyl)-modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises three consecutive LNAs at the first three nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises three consecutive deoxyribonucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.

在某些實施例中，該引導 RNA 分子的 3' 端 (第「n」個) 核苷酸包括選自由以下所組成之群組的修飾：(a) 經 2'-氟修飾的核苷酸、(b) 經 2'-O-甲基修飾的核苷酸、(c) 經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、(d) 鎖核酸 (LNA)、(e) 去氧核糖核苷酸，以及 (a) 至 (e) 中之兩者或更多者之組合。在某些實施例中，該引導 RNA 分子的第 n 個核苷酸包含經 2'-氟修飾的核苷酸。在某些實施例中，該引導 RNA 分子的第 n 個核苷酸包含經 2'-O-甲基修飾的核苷酸。在某些實施例中，該引導 RNA 分子的第 n 個核苷酸包含經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，該引導 RNA 分子的第 n 個核苷酸包含 LNA。在某些實施例中，該引導 RNA 分子的第 n 個核苷酸包含去氧核糖核苷酸。In certain embodiments, the 3' ("nth") nucleotide of the guide RNA molecule comprises a modification selected from the group consisting of: (a) a 2'-fluoro modified nucleotide, (b) a 2'-O-methyl modified nucleotide, (c) a 2'-O-(2-methoxyethyl) modified nucleotide, (d) a locked nucleic acid (LNA), (e) a deoxyribonucleotide, and a combination of two or more of (a) to (e). In certain embodiments, the nth nucleotide of the guide RNA molecule comprises a 2'-fluoro modified nucleotide. In certain embodiments, the nth nucleotide of the guide RNA molecule comprises a 2'-O-methyl modified nucleotide. In certain embodiments, the nth nucleotide of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl) modified nucleotide. In certain embodiments, the nth nucleotide of the guide RNA molecule comprises an LNA. In certain embodiments, the nth nucleotide of the guide RNA molecule comprises a deoxyribonucleotide.

在某些實施例中，該引導 RNA 分子的 3' 端處的 (i) 第 n-1 個核苷酸、(ii) 第 n-2 個核苷酸、(iii) 第 n-3 個核苷酸、(iv) 第 n-4 個核苷酸、(v) 第 n 個與第 n-1 個核苷酸、(vi) 第 n 個與第 n-2 個核苷酸、(vii) 第 n-1 個與第 n-2 個核苷酸、(viii) 第 n 個、第 n-1 個與第 n-2 個核苷酸、(ix) 第 n-1 個、第 n-2 個與第 n-3 個核苷酸、(x) 第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸、或 (xi) 第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自包含選自由以下所組成之群組的修飾：(a) 經 2'-氟修飾的核苷酸、(b) 經 2'-O-甲基修飾的核苷酸、(c) 經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、(d) 鎖核酸 (LNA)、(e) 去氧核糖核苷酸，以及 (a) 至 (e) 中之兩者或更多者之組合。在某些實施例中，該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-氟修飾的核苷酸。在某些實施例中，該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-O-甲基修飾的核苷酸。在某些實施例中，該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為 LNA。在某些實施例中，該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為去氧核糖核苷酸。In certain embodiments, (i) the n-1 nucleotide, (ii) the n-2 nucleotide, (iii) the n-3 nucleotide, (iv) the n-4 nucleotide, (v) the n-1 nucleotide, (vi) the n-2 nucleotide, (vii) the n-1 nucleotide and the n-2 nucleotide, (viii) the n-1 nucleotide and the n-2 nucleotide, (ix) the n-1 nucleotide, the n-2 nucleotide and the n-3 nucleotide, (x) the n-1 nucleotide, the n-2 nucleotide and the n-3 nucleotide, or (xi) the n-1 nucleotide, the n-2 nucleotide, the n-3 nucleotide and the n-4 nucleotide at the 3' end of the guide RNA molecule. Each of the nucleotides comprises a modification selected from the group consisting of: (a) a 2'-fluoro-modified nucleotide, (b) a 2'-O-methyl-modified nucleotide, (c) a 2'-O-(2-methoxyethyl)-modified nucleotide, (d) a locked nucleic acid (LNA), (e) a deoxyribonucleotide, and a combination of two or more of (a) to (e). In certain embodiments, the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-fluorine-modified nucleotides. In certain embodiments, the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-O-methyl modified nucleotides. In certain embodiments, the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-O-(2-methoxyethyl) modified nucleotides. In certain embodiments, the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each LNAs. In certain embodiments, the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each deoxyribonucleotides.

本揭露進一步提供核酸，其包括編碼本文所揭示之引導 RNA 分子的多核苷酸。在某些實施例中，該核酸進一步包括編碼 RNA 引導的核酸酶的多核苷酸。在某些實施例中，RNA 引導的核酸酶為 Cas 蛋白。在某些實施例中，該 Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。本揭露提供載體，其包括本文所揭示之核酸。The disclosure further provides nucleic acids comprising polynucleotides encoding guide RNA molecules disclosed herein. In certain embodiments, the nucleic acids further comprise polynucleotides encoding RNA-guided nucleases. In certain embodiments, the RNA-guided nucleases are Cas proteins. In certain embodiments, the Cas proteins are selected from the group consisting of Cas9, Cas12, Cas13, and combinations thereof. The disclosure provides vectors comprising nucleic acids disclosed herein.

本揭露提供組成物，其包括本文所揭示之引導 RNA 分子。在某些實施例中，本揭露之組成物包括本文所揭示之載體。在某些實施例中，該組成物進一步包括 RNA 引導的核酸酶。替代地或另外，該組成物進一步包括編碼 RNA 引導的核酸酶的核酸。在某些實施例中，RNA 引導的核酸酶為 Cas 蛋白，例如 Cas9、Cas12 及/或 Cas13。在某些實施例中，該組成物包括本文所揭示之核酸，例如，包括編碼引導 RNA 分子的多核苷酸的核酸。The present disclosure provides compositions comprising guide RNA molecules disclosed herein. In certain embodiments, the compositions disclosed herein comprise vectors disclosed herein. In certain embodiments, the composition further comprises an RNA-guided nuclease. Alternatively or additionally, the composition further comprises a nucleic acid encoding the RNA-guided nuclease. In certain embodiments, the RNA-guided nuclease is a Cas protein, such as Cas9, Cas12, and/or Cas13. In certain embodiments, the composition comprises a nucleic acid disclosed herein, for example, a nucleic acid comprising a polynucleotide encoding a guide RNA molecule.

本揭露進一步包括一種核糖核酸蛋白 (RNP) 複合物，其包括本揭露之經修飾的引導 RNA 分子以及 RNA 引導的核酸酶。在某些實施例中，RNA 引導的核酸酶為 Cas 蛋白。在某些實施例中，該 Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。The present disclosure further includes a ribonucleoprotein (RNP) complex comprising a modified guide RNA molecule of the present disclosure and an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of: Cas9, Cas12, Cas13, and combinations thereof.

本揭露進一步包括一種細胞，其包含本文所揭示之組成物或 RNP 複合物。The disclosure further includes a cell comprising the composition or RNP complex disclosed herein.

本揭露進一步提供一種修飾細胞之方法。在某些實施例中，該方法包括使細胞與本文所揭示之組成物或 RNP 複合物接觸。在某些實施例中，該接觸包括藉由電穿孔將組成物引入細胞中。The present disclosure further provides a method for modifying cells. In certain embodiments, the method comprises contacting the cell with a composition or RNP complex disclosed herein. In certain embodiments, the contacting comprises introducing the composition into the cell by electroporation.

本揭露提供治療有需要之個體之方法。在某些實施例中，該方法包括藉由使個體的細胞與本文所揭示之組成物或 RNP 複合物接觸，離體修飾該細胞，以及將經修飾的細胞送回至該個體。The present disclosure provides methods for treating an individual in need thereof. In certain embodiments, the method comprises ex vivo modifying a cell of the individual by contacting the cell with a composition or RNP complex disclosed herein, and returning the modified cell to the individual.

本揭露進一步提供基因編輯系統，其包含一或多個本揭露之經修飾的引導 RNA，本文所揭示之一或多個核酸、一或多個載體、一或多個組成物及/或一或多個 RNP 複合物。The present disclosure further provides a gene editing system, which comprises one or more modified guide RNAs of the present disclosure, one or more nucleic acids, one or more vectors, one or more compositions and/or one or more RNP complexes disclosed herein.

相關申請的交叉引用Cross-references to related applications

本申請案主張 2023 年 4 月 7 日提交的美國臨時申請案第 63/458,017 號之優先權，該申請案之內容以引用方式全文併入本文中。This application claims priority to U.S. Provisional Application No. 63/458,017, filed on April 7, 2023, the contents of which are incorporated herein by reference in their entirety.

本揭露涉及經修飾之引導 RNA分子、包括此類經修飾之引導 RNA 的醫藥組成物以及藉由投予經修飾之引導 RNA 分子來修飾細胞的方法。本揭露部分基於以下發現：將二硫代磷酸酯鍵結引入 gRNA 分子中會消除由硫代磷酸酯鍵結產生之對掌性中心，從而產生穩定的 gRNA 分子、在此等經修飾的磷酸位置處不包括非鏡像異構物之 gRNA 組成物以及經更好定義之 gRNA 群體。如圖37、圖 38、圖 57 及圖 58 中所示，與不包括二硫代磷酸酯鍵結之 gRNA 分子相比，在強制降解條件下 (諸如在鹼性應激條件及酸性應激條件下)，在 5' 及/或 3' 端處包括二硫代磷酸酯鍵結之 gRNA 分子導致穩定性增加。另外，如圖61、圖 65 及圖 69 中所示，與不包括二硫代磷酸酯鍵結之 gRNA 分子相比，在 5' 及/或 3' 端處包括二硫代磷酸酯鍵結之 gRNA 分子具有相當或增加的編輯效率。The present disclosure relates to modified guide RNA molecules, pharmaceutical compositions comprising such modified guide RNAs, and methods of modifying cells by administering modified guide RNA molecules. The present disclosure is based in part on the discovery that the introduction of phosphorodithioate bonds into gRNA molecules eliminates the chiral center created by the phosphorothioate bonds, thereby generating stable gRNA molecules, gRNA compositions that do not include non-mirror image isomers at such modified phosphate positions, and better defined gRNA populations. As shown in Figures 37, 38, 57 and 58, gRNA molecules including dithiophosphate bonds at the 5' and/or 3' ends resulted in increased stability under forced degradation conditions (such as under alkaline stress conditions and acidic stress conditions) compared to gRNA molecules that do not include dithiophosphate bonds. In addition, as shown in Figures 61, 65 and 69, gRNA molecules including dithiophosphate bonds at the 5' and/or 3' ends have comparable or increased editing efficiencies compared to gRNA molecules that do not include dithiophosphate bonds.

為清楚起見，但不作為限制，將本發明所揭示之標的之詳細描述分為以下小節： I.         定義； II.       經修飾之引導 RNA； III.      RNA 引導的核酸酶； IV.      組成物； V.       使用方法； VI.      基因編輯系統；及 VII.     例示性實施例。I.定義For the sake of clarity, but not as a limitation, the detailed description of the subject matter disclosed in the present invention is divided into the following subsections: I. Definitions; II. Modified Guide RNAs; III. RNA-Guided Nucleases; IV. Compositions; V. Methods of Use; VI. Gene Editing Systems; and VII. Exemplary Embodiments.I.Definitions

除非另有定義，否則本文所用之全部技術及科學術語具有與一般熟習本揭露之主題所屬技術者通常所瞭解之含義相同的含義。下列參考文獻提供技術人員本揭露中所使用的許多術語的一般定義：Singleton 等人，Dictionary of Microbiology and Molecular Biology (第 2 版，1994)；The Cambridge Dictionary of Science and Technology (Walker 編，1988)；The Glossary of Genetics，第 5 版，R. Rieger 等人(編)，Springer Verlag (1991)；以及 Hale & Marham, The Harper Collins Dictionary of Biology (1991)。如本文所用，除非另有說明，否則以下術語具有以下賦予其等之含義。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the subject matter of this disclosure belongs. The following references provide general definitions of many of the terms used in this disclosure by those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise specified, the following terms have the meanings assigned to them below.

如本文所用，當「一」或「一種」一詞的使用與請求項及/或說明書中的術語「包含」結合使用時，其可意指「一個」，但亦與「一個或多個」、「至少一個」和「一個或大於一個」的含義一致。As used herein, when the terms "a" or "an" are used in conjunction with the term "comprising" in the claims and/or the specification, they may mean "one", but are also consistent with the meanings of "one or more", "at least one", and "one or more than one".

術語「約」或「大約」意指特定值處於本技術領域中具有通常知識者所判定之可接受的誤差範圍內，其部分地取決於如何測量或判定該值，即，取決於測量系統的局限性。例如，按照本技術領域中的實務，「約」可意指 3 倍或 3 倍以上的標準偏差。可替代地，「約」可意指給定值的至多 20%、最佳至多 10%、更佳至多 5% 並且更佳至多 1% 的範圍。可替代地，特別是關於生物系統或過程，該術語意指數值的一個數量級內，最佳地在數值的 5 倍以內，並且更佳地在數值的 2 倍以內。The term "about" or "approximately" means that a particular value is within an acceptable range of error as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined,i.e. , on the limitations of the measurement system. For example, according to practice in the art, "about" may mean 3 or more times the standard deviation. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term means within an order of magnitude of a value, preferably within 5 times, and more preferably within 2 times of a value.

如本文所用，術語「包含」、「包括」、「具有 (having、has)」、「可」、「含有」及其變異體意欲為不排除其他行為或結構之開放式連接詞 (open-ended transitional phrase)、術語、或字。本揭露亦涵蓋「包含」本文所呈現之實施例或元件、「由其組成」及「基本上由其組成」之本文所呈現之實施例或元件，無論是否明確陳述。As used herein, the terms "comprise", "include", "having", "has", "may", "contain" and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude other actions or structures. The present disclosure also encompasses embodiments or elements presented herein that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

術語「偶合」可指兩個或更多個組分藉由相互作用、鍵合、連接、力或繫帶連接或聯合，以便將兩個或更多個組分保持在一起。在某些實施例中，術語「偶合」涵蓋直接或間接結合，其中例如第一組分直接與第二組分結合，或者一個或多個中間分子設置於第一組分與第二組分之間。示例性鍵包含共價鍵、離子鍵、凡得瓦 (van der Waals) 交互作用及技術人員可識別的其他鍵。The term "coupled" may refer to two or more components being connected or joined by an interaction, bond, connection, force or tie so as to hold the two or more components together. In certain embodiments, the term "coupled" encompasses direct or indirect binding, where, for example, a first component is directly bound to a second component, or one or more intermediary molecules are disposed between the first component and the second component. Exemplary bonds include covalent bonds, ionic bonds, van der Waals interactions, and other bonds recognizable to a skilled artisan.

如本文所用，術語「檢測 (detect)」或「檢測 (detection)」指示在空間之有限部分，包括但不限於樣品中判定標靶 (例如，核酸標靶) 之存在性 (existence) 及/或存在情況 (presence)。如本文所用，術語「檢測 (detect)」或「檢測 (detection)」可包含判定標靶的化學及/或生物學特性，包括但不限於相互作用的能力，特定而言結合其他化合物的能力，激活另一化合物及技術人員在閱讀本揭露內容後可識別的額外特性的能力。檢測可為定量的或定性的。當檢測指代、涉及或關於標靶或信號的數量或量的測量 (也稱為定量) 時，檢測為「定量的」，包括但不限於經設計用於判定標靶或信號的量或比例的任何分析。當檢測指代、涉及或關於根據相對於另一未量化的標靶或信號的相對豐度來識別目標或信號的質量或種類時，檢測是「定性的」。As used herein, the term "detect" or "detection" indicates determining the existence and/or presence of a target (e.g., a nucleic acid target) in a limited portion of space, including but not limited to a sample. As used herein, the term "detect" or "detection" may include determining the chemical and/or biological properties of a target, including but not limited to the ability to interact, particularly the ability to bind to other compounds, the ability to activate another compound, and additional properties that are discernible to a skilled artisan after reading this disclosure. Detection may be quantitative or qualitative. Detection is "quantitative" when it refers to, involves, or relates to the measurement of the amount or quantity of a target or signal (also referred to as quantification), including but not limited to any analysis designed to determine the amount or ratio of a target or signal. A test is "qualitative" when it refers to, involves, or is concerned with identifying the quality or kind of a target or signal based on its relative abundance to another, unquantified target or signal.

如本文所用，術語「域」係指蛋白質或核酸 (例如，gRNA 分子) 之區段。除非另外指示，否則域不需要具有任何特定的功能特性。As used herein, the term "domain" refers to a segment of a protein or nucleic acid (e.g., a gRNAmolecule). Unless otherwise indicated, a domain is not required to have any particular functional properties.

如本文所用，術語「編輯效率」係指在所關註標靶區域中插入或缺失核苷酸之序列讀數總數與在藉由 RNA 引導的核酸酶裂解之後的序列讀數總數之比。As used herein, the term "editing efficiency" refers to the ratio of the total number of sequence reads with nucleotide insertions or deletions in the annotated target region of interest to the total number of sequence reads after cleavage by RNA-guided nuclease.

如本文可互換使用，術語「引導 RNA」、「gRNA」或「gRNA 分子」係指促進 RNA 引導的核酸酶特異性靶向或歸巢至標靶核酸的核酸。As used interchangeably herein, the terms "guide RNA," "gRNA," or "gRNA molecule" refer to a nucleic acid that promotes specific targeting or homing of an RNA-guided nuclease to a target nucleic acid.

如本文所用，術語「雜交」係指兩個單股多核苷酸非共價結合以形成穩定的雙股多核苷酸的方法。As used herein, the term "hybridization" refers to the process by which two single-stranded polynucleotides non-covalently associate to form a stable double-stranded polynucleotide.

如本文所用，術語「受試者」或「個體」係指脊椎動物或無脊椎動物，諸如人類或非人類動物，例如哺乳動物。哺乳動物包括但不限於人類、非人類靈長類動物、農場動物、競賽動物、囓齒動物和寵物。非人類動物個體的非限制性實例包括囓齒動物，諸如小鼠、大鼠、倉鼠、天竺鼠、兔、狗、貓、綿羊、豬、山羊、牛、馬、猿及猴。在某些實施例中，受試者或個體為人類。As used herein, the term "subject" or "individual" refers to a vertebrate or invertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, game animals, rodents, and pets. Non-limiting examples of non-human animal individuals include rodents, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, apes, and monkeys. In certain embodiments, the subject or individual is a human.

如本文所用，術語「活體外」涉及人工環境及在人工環境內發生的過程或反應。活體外環境例如為試管和細胞培養物，但不以此為限。As used herein, the term "in vitro " refers to an artificial environment and processes or reactions that occur within an artificial environment. Examples ofin vitro environments include, but are not limited to, test tubes and cell cultures.

如本文所用，術語「活體內」涉及自然環境 (例如，動物或細胞) 及在自然環境中發生的過程或反應，諸如胚胎發育、細胞分化、神經管形成等。As used herein, the term "in vivo " refers to the natural environment (eg , animals or cells) and processes or reactions that occur in the natural environment, such as embryonic development, cell differentiation, neural tube formation, etc.

如本文所用，「標記」係指允許直接或間接偵測的藥劑。標記包括但不限於螢光組成物、顯色標記、電子緻密標記、化學發光標記及放射性標記。標記之非限制性實例包括綠色螢光蛋白 (「GFP」)、mCherry、dtTomato 或此項技術中已知之其他螢光蛋白 (例如，Shaner 等人, A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005) (藉由引用併入本文)，³²P、¹⁴C、¹²⁵I、³H 及¹³¹I、螢光團 (諸如稀土螯合物或螢光黃及其衍生物)、玫瑰紅 (Rhodamine/rhodamine) 及其衍生物，丹磺醯 (dansyl)，繖形酮 (umbelliferone)、螢光素酶 (諸如螢火蟲螢光素酶及細菌螢光素純酶) (美國專利第 4,737,456 號)、螢光素、2,3-二氫呔𠯤二酮，以及產生可偵測訊號之酶，例如山葵過氧化酶 (horseradish peroxidase, HRP)、鹼性磷酸酶 (alkaline phosphorus sour enzyme)、β 半乳糖苷酶、葡萄糖澱粉酶、溶菌酶、碳水化合物氧化酶 (諸如葡萄糖氧化酶、半乳糖氧化酶及葡萄糖-6-磷酸去氫酶 (glucose-6-phosphate dehydrogenase，G6PD)) 及雜環氧化酶 (諸如尿酸酶及黃嘌呤氧化酶)。As used herein, "label" refers to an agent that allows direct or indirect detection. Labels include, but are not limited to, fluorescent compositions, chromogenic labels, electronically precise labels, chemiluminescent labels, and radioactive labels. Non-limiting examples of labels include green fluorescent protein ("GFP"), mCherry, dtTomato or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005) (incorporated herein by reference),³² P,¹⁴ C,¹²⁵ I,³ H and¹³¹ I, fluorophores (such as rare earth chelates or fluorescent yellow and its derivatives), rhodamine/rhodamine and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and bacterial luciferin pure enzyme) (U.S. Patent No. 4,737,456 ), fluorescein, 2,3-dihydropyridine-1,3-dione, and enzymes that generate detectable signals, such as horseradish peroxidase (HRP), alkaline phosphorus sour enzyme, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)) and heterocyclic oxidases (such as uricase and xanthine oxidase).

術語「核酸」或「多核苷酸」包括任何包含核苷酸聚合物的化合物及/或物質。每個核苷酸由鹼基具體而言嘌呤或嘧啶鹼基 (即，胞嘧啶 (C)、鳥嘌呤 (G)、腺嘌呤 (A)、胸腺嘧啶 (T) 或尿嘧啶 (U))、糖 (即，去氧核糖或核糖) 及磷酸基團構成。通常，核酸分子通過鹼基序列進行描述，其中該等鹼基代表核酸分子的一級結構 (線性結構)。鹼基序列通常由 5’ 至 3’ 表示。術語核酸包括：去氧核糖核酸 (DNA)，包括例如，互補 DNA (cDNA) 及基因體 DNA；核糖核酸 (RNA)，例如傳訊 RNA (mRNA)；DNA 或 RNA 的合成形式；以及包含這些分子中之兩者或更多者的混合聚合物。核酸分子可為線性或環狀的。此外，術語核酸包括有義股及反義股兩者，以及單股及雙股形式。此外，本文所述之核酸可含有天然存在或非天然存在之核苷酸。非天然存在之核苷酸的實例包括帶有衍生醣、磷酸鹽鍵結或化學修飾殘基的經修飾之核苷酸鹼基。The term "nucleic acid" or "polynucleotide" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a base sequence, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented from 5' to 3'. The term nucleic acid includes: deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), such as messaging RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. Furthermore, the term nucleic acid includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acids described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars, phosphate linkages, or chemically modified residues.

術語「核苷」係指包括嘌呤或嘧啶鹼基 (即，胞嘧啶 (C)、鳥嘌呤 (G)、腺嘌呤 (A)、胸腺嘧啶 (T) 或尿嘧啶 (U)) 及糖 (即，去氧核糖或核糖) 的化合物。The term "nucleoside" refers to a compound that includes a purine or pyrimidine base(i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)) and a sugar(i.e., deoxyribose or ribose).

「分離的」核酸係指已經與其天然環境的組分分離的核酸分子。分離的核酸包括通常包含核酸分子之細胞中所含之核酸分子，但是核酸分子存在於染色體外或與自然染色體位置不同之染色體位置。An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from the natural chromosomal location.

術語「複數」係指大於一個的數。在某些實施例中，術語「複數個引導 RNA」係指大於一個的引導 RNA 數量。例如，但不作為限制，複數個引導 RNA 包括至少兩個引導 RNA。在某些實施例中，術語「複數種核酸」係指大於一種的核酸數量。例如，但不作為限制，複數種核酸包括至少兩種核酸。The term "plurality" refers to a number greater than one. In certain embodiments, the term "plurality of guide RNAs" refers to a number greater than one guide RNA. For example, but not by way of limitation, a plurality of guide RNAs includes at least two guide RNAs. In certain embodiments, the term "plurality of nucleic acids" refers to a number greater than one nucleic acid. For example, but not by way of limitation, a plurality of nucleic acids includes at least two nucleic acids.

術語「參考分子」或「對照分子」，例如，如本文所用之參考或對照 gRNA 分子，係指與標的分子，例如標的 gRNA 分子比較的分子。例如但不作為限制，標的 gRNA 分子包括與參考分子相比之一或多個修飾，例如，核苷酸修飾。The term "reference molecule" or "control molecule", e.g., a reference or control gRNA molecule as used herein, refers to a molecule to which a target molecule, e.g., a target gRNA molecule, is compared. For example, but not limitation, the target gRNA molecule includes one or more modifications, e.g., nucleotide modifications, compared to the reference molecule.

如本文所用，術語「特異性結合」係指相對於樣品中的其他分子 (例如蛋白質或核酸) 優先結合至標靶分子 (例如蛋白質或核酸)。As used herein, the term "specific binding" refers to preferential binding to a target molecule (e.g., a protein or nucleic acid) relative to other molecules (e.g., proteins or nucleic acids) in a sample.

如本文中所使用的「治療」(及其語法變異體，諸如「治療過程」或「治療中」)，係指試圖改變受治療個體之疾病自然病程的臨床干預，並且可進行預防或在臨床病理過程中執行。期望之治療效果包括但不限於預防疾病之發生或複發、減輕症狀、減輕疾病之任何直接或間接病理後果、預防轉移、降低疾病進展之速度、改善或減輕疾病狀態、以及緩解或改善預後。該降低可為併發症、體徵或症狀之嚴重程度或進展至另一等級的可能性的至少 10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、98% 或 99% 降低。「治療」亦可指抑制癌症之增殖或進展至更高等級至少 10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、98% 或 99%。在某些實施例中，本揭露之 gRNA 用於延遲疾病之發展或減慢疾病之進程。As used herein, "treatment" (and grammatical variations such as "treatment process" or "treating") refers to clinical intervention that attempts to alter the natural course of a disease in the individual being treated, and may be performed preventively or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or reducing the disease state, and relieving or improving prognosis. The reduction may be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% reduction in the severity of complications, signs, or symptoms, or the likelihood of progression to another level. "Treatment" may also refer to inhibiting the proliferation or progression of cancer to a higher level by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%. In certain embodiments, the gRNA disclosed herein is used to delay the development of a disease or slow the progression of a disease.

如本文所用，術語「治療效果」係指由藥理活性物質在個體中引起的局部或全身效果。As used herein, the term "therapeutic effect" refers to a local or systemic effect caused by a pharmacologically active substance in a subject.

如本文所用，術語「治療有效量」及「有效量」係指本揭露之組成物的量，該量以適用於任何醫療之合理效益/風險比在個體之至少細胞亞群中有效產生一些期望的治療效果。As used herein, the terms "therapeutically effective amount" and "effective amount" refer to an amount of a composition of the present disclosure that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment.

術語「變異」當指給定多肽殘基位置處的變異時，係指彼等殘基處的任何修飾。例如但不作為限制，術語「變異」可指用不同的胺基酸取代給定位置處的胺基酸。The term "variant" when referring to a variation at a given polypeptide residue position refers to any modification at that residue. For example, but not by way of limitation, the term "variant" may refer to replacing an amino acid at a given position with a different amino acid.

如本文所用，術語「載體」係指能夠繁殖與其連接的另一核酸的核酸分子。該術語包括作為自我複製核酸結構之載體以及併入已引入該宿主細胞的基因體中的載體。某些載體能夠指導與該等載體可操作地連接的核酸之表現。此類載體在本文中稱為「表現載體」。II.經修飾的引導RNA分子As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell that has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."II.Modified GuideRNAMolecules

本揭露提供包括一或多個修飾的 gRNA。在某些實施例中，與不包括一或多個修飾的 gRNA (例如，對照或參考 gRNA) 相比，一或多個修飾增加了 gRNA 之穩定性。例如但不作為限制，修飾藉由防止 gRNA 分子被核酸酶 (例如核酸內切酶及/或核酸外切酶) 進行降解來改善所揭示之 gRNA 分子的穩定性。此類增強的穩定性可以提高經修飾之 gRNA 分子的治療功效。此外，修飾可以提供更好的保存期穩定性且產生具有更高品質之 gRNA 組成物，此係因為非鏡像異構物不會存在於此類組成物中。該等修飾亦可提高編輯效率且減少可能的脫靶效應。The present disclosure provides gRNAs comprising one or more modifications. In certain embodiments, one or more modifications increase the stability of the gRNA compared to a gRNA that does not include the one or more modifications (e.g., a control or reference gRNA). For example, but not by way of limitation, the modifications improve the stability of the disclosed gRNA molecules by preventing the gRNA molecules from being degraded by nucleases (e.g., endonucleases and/or exonucleases). Such enhanced stability can improve the therapeutic efficacy of the modified gRNA molecules. In addition, the modifications can provide better shelf-life stability and produce gRNA compositions with higher quality because non-mirror isomers are not present in such compositions. Such modifications can also improve editing efficiency and reduce potential off-target effects.

在某些實施例中，與參考 gRNA 相比，在降解條件 (例如，鹼性應激條件及/或酸性應激條件) 下，本揭露之經修飾之 gRNA 表現出約 1% 或更多，例如約 2% 或更多、約 3% 或更多、約 4% 或更多、約 5% 或更多、約 6% 或更多、約 7% 或更多、約 8% 或更多、約 9% 或更多、約 10% 或更多、約 15% 或更多、約 20% 或更多、約 25% 或更多、約 30% 或更多、約 35% 或更多、約 40% 或更多、約 45% 或更多、約 50% 或更多或約 55% 或更多的穩定性增加。在某些實施例中，與參考 gRNA 相比，在降解條件 (例如，鹼性應激條件及/或酸性應激條件) 下，本揭露之經修飾之 gRNA 表現出約 1% 至約 60% 的穩定性增加。在某些實施例中，本揭露之經修飾之 gRNA 在酸性應激條件下表現出約 1% 或更多、約 2% 或更多、約 3% 或更多、約 4% 或更多或約 5% 或更多的穩定性增加。在某些實施例中，與參考 gRNA 相比，在酸性應激條件下，本揭露之經修飾之 gRNA 表現出約 1% 至約 5% 的穩定性增加。在某些實施例中，本揭露之經修飾之 gRNA 在鹼性應激條件下表現出約 5% 或更多、約 10% 或更多、約 15% 或更多、約 20% 或更多、約 25% 或更多、約 30% 或更多、約 35% 或更多、約 40% 或更多、約 45% 或更多、約 50% 或更多或約 55% 或更多的穩定性增加。在某些實施例中，與參考 gRNA 相比，在鹼性應激條件下，本揭露之經修飾之 gRNA 表現出約 1% 至約 60% 的穩定性增加。在某些實施例中，與參考 gRNA 相比，在降解條件 (例如，鹼性應激條件及/或酸性應激條件) 下，本揭露之經修飾之 gRNA 表現出約 1% 或更多，例如約 2% 或更多、約 3% 或更多、約 4% 或更多、約 5% 或更多、約 6% 或更多、約 7% 或更多、約 8% 或更多、約 9% 或更多、約 10% 或更多、約 15% 或更多、約 20% 或更多、約 25% 或更多、約 30% 或更多、約 35% 或更多、約 40% 或更多、約 45% 或更多、約 50% 或更多或約 55% 或更多的降解減少。在某些實施例中，與參考 gRNA 相比，在降解條件 (例如，鹼性應激條件及/或酸性應激條件) 下，本揭露之經修飾之 gRNA 表現出約 1% 至約 60% 的降解減少。在某些實施例中，本揭露之經修飾之 gRNA 在酸性應激條件下表現出約 1% 或更多、約 2% 或更多、約 3% 或更多、約 4% 或更多或約 5% 或更多的降解減少。在某些實施例中，與參考 gRNA 相比，在酸性應激條件下，本揭露之經修飾之 gRNA 表現出約 1% 至約 5% 的降解減少。在某些實施例中，本揭露之經修飾之 gRNA 在鹼性應激條件下表現出約 5% 或更多、約 10% 或更多、約 15% 或更多、約 20% 或更多、約 25% 或更多、約 30% 或更多、約 35% 或更多、約 40% 或更多、約 45% 或更多、約 50% 或更多或約 55% 或更多的降解減少。在某些實施例中，與參考 gRNA 相比，在鹼性應激條件下，本揭露之經修飾之 gRNA 表現出約 1% 至約 60% 的降解減少。在某些實施例中，在經受降解條件後約 24 小時、48 小時、3 天、4 天、5 天、6 天或 7 天時觀察到穩定性之增加及/或降解之減少。在某些實施例中，熱應激條件包括使 gRNA 經受高溫，例如，高於約 37℃ 之溫度。在某些實施例中，氧化應激條件包括使 gRNA 經受自由基及/或化合物 (例如，H₂O₂)。在某些實施例中，鹼性應激條件包括使 gRNA 經受鹼性 pH，例如，大於約 8，例如，約 11 的 pH。在某些實施例中，酸性應激條件包括使 gRNA 經受酸性 pH，例如，小於約 6，例如，約 5 的 pH。在某些實施例中，參考 gRNA 可為具有與經修飾之 gRNA 相同的核苷酸序列之 gRNA (例如，沒有與經修飾之 gRNA 相同的修飾)。在某些實施例中，參考 gRNA 可為具有靶向域的 gRNA，該靶向域具有與經修飾之 gRNA 相同的序列 (例如，沒有與經修飾之 gRNA 相同的修飾)。在某些實施例中，參考 gRNA 可為具有與經修飾之 gRNA 相同數量之核苷酸的 gRNA (例如，沒有與經修飾之 gRNA 相同的修飾)。In certain embodiments, the modified gRNA of the present disclosure exhibits an increase in stability of about 1% or more, such as about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, or about 55% or more under degradation conditions (e.g., alkaline stress conditions and/or acidic stress conditions) compared to a reference gRNA. In certain embodiments, compared to a reference gRNA, under degradation conditions (e.g., alkaline stress conditions and/or acidic stress conditions), the modified gRNA of the present disclosure exhibits an increase in stability of about 1% to about 60%. In certain embodiments, the modified gRNA of the present disclosure exhibits an increase in stability of about 1% or more, about 2% or more, about 3% or more, about 4% or more, or about 5% or more under acidic stress conditions. In certain embodiments, compared to a reference gRNA, under acidic stress conditions, the modified gRNA of the present disclosure exhibits an increase in stability of about 1% to about 5%. In certain embodiments, the modified gRNA of the present disclosure exhibits an increase in stability of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, or about 55% or more under alkaline stress conditions. In certain embodiments, compared to a reference gRNA, under alkaline stress conditions, the modified gRNA of the present disclosure exhibits an increase in stability of about 1% to about 60%. In certain embodiments, the modified gRNA of the present disclosure exhibits about 1% or more, such as about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, or about 55% or more degradation reduction under degradation conditions (e.g., alkaline stress conditions and/or acidic stress conditions) compared to a reference gRNA. In certain embodiments, the modified gRNA of the present disclosure exhibits about 1% to about 60% reduction in degradation under degradation conditions (e.g., alkaline stress conditions and/or acidic stress conditions) compared to a reference gRNA. In certain embodiments, the modified gRNA of the present disclosure exhibits about 1% or more, about 2% or more, about 3% or more, about 4% or more, or about 5% or more reduction in degradation under acidic stress conditions. In certain embodiments, the modified gRNA of the present disclosure exhibits about 1% to about 5% reduction in degradation under acidic stress conditions compared to a reference gRNA. In certain embodiments, the modified gRNA of the present disclosure exhibits about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, or about 55% or more degradation reduction under alkaline stress conditions. In certain embodiments, the modified gRNA of the present disclosure exhibits about 1% to about 60% degradation reduction under alkaline stress conditions compared to a reference gRNA. In certain embodiments, an increase in stability and/or a decrease in degradation is observed at about 24 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, or 7 days after being subjected to degradation conditions. In certain embodiments, heat stress conditions include subjecting the gRNA to high temperatures, e.g., temperatures above about 37° C. In certain embodiments, oxidative stress conditions include subjecting the gRNA to free radicals and/or compounds (e.g., H₂ O₂ ). In certain embodiments, alkaline stress conditions include subjecting the gRNA to an alkaline pH, e.g., a pH greater than about 8, e.g., about 11. In certain embodiments, acidic stress conditions include subjecting the gRNA to an acidic pH, e.g., a pH less than about 6, e.g., about 5. In certain embodiments, the reference gRNA may be a gRNA having the same nucleotide sequence as the modified gRNA (e.g., without the same modifications as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA having a targeting domain that has the same sequence as the modified gRNA (e.g., without the same modifications as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA having the same number of nucleotides as the modified gRNA (e.g., without the same modifications as the modified gRNA).

在某些實施例中，本揭露之 gRNA 包括一或多個、兩個或更多個、三個或更多個、四個或更多個、五個或更多個、六個或更多個、七個或更多個、八個或更多個、九個或更多個、十個或更多個、十一個或更多個、十二個或更多個、十三個或更多個、十四個或更多個、或十五個或更多個修飾。在某些實施例中，本揭露之 gRNA 包括至少兩個、至少三個、至少四個、至少五個、至少六個、至少七個、至少八個、至少九個、至少十個核苷酸修飾、至少十一個核苷酸修飾、至少十二個核苷酸修飾、至少十三個核苷酸修飾、至少十四個核苷酸修飾或至少十五個核苷酸修飾。在某些實施例中，本揭露之 gRNA 包括至少約十一個修飾。In certain embodiments, the gRNA disclosed herein comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more modifications. In certain embodiments, the gRNA disclosed herein comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten nucleotide modifications, at least eleven nucleotide modifications, at least twelve nucleotide modifications, at least thirteen nucleotide modifications, at least fourteen nucleotide modifications, or at least fifteen nucleotide modifications. In certain embodiments, the gRNA disclosed herein comprises at least about eleven modifications.

在某些實施例中，本揭露之 gRNA 的長度為約 20 至約 200 個核苷酸，例如約 20 至約 190、約 20 至約 180、約 20 至約 170、約 20 至約 160、約 20 至約 150、約 20 至約 140、約 20 至約 130、約 20 至約 120、約 20 至約 110、約 20 至約 100、約 30 至約 200、約 40 至約 200、約 50 至約 200、約 60 至約 200、約 70 至約 200、約 80 至約 200、約 90 至約 200、約 50 至約 150、約 80 至約 120、或約 90 至約 100 個核苷酸。在某些實施例中，本揭露之 gRNA 的長度為約 80 至約 120 個核苷酸。在某些實施例中，本揭露之 gRNA 的長度為約 5、約 10、約 15、約 20、約 25、約 30、約 35、約 40、約 45、約 50、約 55、約 60、約 65、約 70、約 75、約 80、約 85、約 90、約 95、約 100、約 105、約 110、約 115、約 120、約 125、約 130、約 135、約 140、約 145、約 150、約 155、約 160、約 165、約 170、約 175、約 180、約 185、約 190、約 195、約 200 或更多個核苷酸。In certain embodiments, the gRNA disclosed herein has a length of about 20 to about 200 nucleotides, such as about 20 to about 190, about 20 to about 180, about 20 to about 170, about 20 to about 160, about 20 to about 150, about 20 to about 140, about 20 to about 130, about 20 to about 120, about 20 to about 110, about 20 to about 100, about 30 to about 200, about 40 to about 200, about 50 to about 200, about 60 to about 200, about 70 to about 200, about 80 to about 200, about 90 to about 200, about 50 to about 200, or about 100 to about 150. In some embodiments, the gRNA of the present disclosure has a length of about 80 to about 120 nucleotides. In certain embodiments, the gRNA disclosed herein has a length of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200 or more nucleotides.

在某些實施例中，本揭露之 gRNA 中存在之約 1% 至約 20% 的核苷酸被修飾，例如，本揭露之 gRNA 中存在之約 1% 至約 19%、約 1% 至約 18%、約 1% 至約 17%、約 1% 至約 16%、約 1% 至約 15%、約 1% 至約 14%、約 1% 至約 13%、約 1% 至約 12%、約 1% 至約 11%、約 1% 至約 10%、約 1% 至約 9%、約 1% 至約 8%、約 1% 至約 7%、約 1% 至約 6%、約 1% 至約 5%、約 1% 至約 4%、約 1% 至約 3%、約 1% 至約 2%、約 2% 至約 20%、約 3% 至約 20%、約 4% 至約 20%、約 5% 至約 20%、約 6% 至約 20%、約 7% 至約 20%、約 8% 至約 20%、約 9% 至約 20%、約 10% 至約 20%、約 11% 至約 20%、約 12% 至約 20%、約 13% 至約 20%、約 14% 至約 20% 、約 15% 至約 20%、約 16% 至約 20%、約 17% 至約 20%、約 18% 至約 20%、約 19% 至約 20%、約 5% 至約 15% 或約 10% 至約 15% 的核苷酸被修飾。在某些實施例中，本揭露之 gRNA 中存在之約 1% 至約 15% 的核苷酸被修飾。在某些實施例中，本揭露之 gRNA 中存在之約 1% 至約 10% 的核苷酸被修飾。在某些實施例中，本揭露之 gRNA 中存在之約 1% 至約 6% 的核苷酸被修飾。在某些實施例中，gRNA 中存在之約 1%、約 2%、約 3%、約 4%、約 5%、約 6%、約 7%、約 8%、約 9%、約 10%、約 11%、約 12%、約 13%、約 14%、約 15%、約 16%、約 17%、約 18%、約 19% 或約 20% 或更多的核苷酸被修飾。In certain embodiments, about 1% to about 20% of the nucleotides present in the gRNA of the present disclosure are modified, for example, about 1% to about 19%, about 1% to about 18%, about 1% to about 17%, about 1% to about 16%, about 1% to about 15%, about 1% to about 14%, about 1% to about 13%, about 1% to about 12%, about 1% to about 11%, about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2 ... About 20% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 11% to about 20%, about 12% to about 20%, about 13% to about 20%, about 14% to about 20%, about 15% to about 20%, about 16% to about 20%, about 17% to about 20%, about 18% to about 20%, about 19% to about 20%, about 5% to about 15%, or about 10% to about 15% of the nucleotides are modified. In certain embodiments, about 1% to about 15% of the nucleotides present in the gRNA of the present disclosure are modified. In certain embodiments, about 1% to about 10% of the nucleotides present in the gRNA of the present disclosure are modified. In certain embodiments, about 1% to about 6% of the nucleotides present in the gRNA of the present disclosure are modified. In certain embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more of the nucleotides present in the gRNA are modified.

在某些實施例中，本揭露之 gRNA 分子具有圖1 中所示之結構。例如但不作為限制，本揭露之 gRNA 分子在其 5' 端處包括間隔區 (圖1 中的藍色區域)。本揭露之 gRNA 分子的額外例示性結構提供於Daniel 等人, Frontiers in Genome Editing 2:617910 (2021) 之圖 1A 中，該文獻之內容藉由引用全文併入本文 (間隔區在Daniel 等人之圖 1A 中以藍色及紅色標註)。In certain embodiments, the gRNA molecule disclosed herein has the structure shown in Figure 1. For example, but not by way of limitation, the gRNA molecule disclosed herein includes a spacer at its 5' end (the blue region in Figure 1). Additional exemplary structures of gRNA molecules disclosed herein are provided in Figure 1A of Daniel et al., Frontiers in Genome Editing 2:617910 (2021), the contents of which are incorporated herein by reference in their entirety (the spacer is annotated in blue and red in Figure 1A of Daniel et al.).

在某些實施例中，本揭露之 gRNA 分子在 gRNA 分子之間隔區中包括一或多個修飾。在某些實施例中，本發明所揭示之 gRNA 分子的間隔區與標靶核酸之序列互補，例如，至少約 80%、約 85%、約 90%、95%、約 98%、約 99% 或約 100% 互補。在某些實施例中，間隔區包括存在於 gRNA 分子之 5' 端處的前 30 個核苷酸，例如前 29 個核苷酸、前 28 個核苷酸、前 27 個核苷酸、前 26 個核苷酸、前 25 個核苷酸、前 24 個核苷酸、前 23 個核苷酸、前 22 個核苷酸、前 21 個核苷酸、前 20 個核苷酸、前 19 個核苷酸、前 18 個核苷酸、前 17 個核苷酸、前 16 個核苷酸或前 15 個核苷酸。在某些實施例中，間隔區包括存在於 gRNA 分子之 5' 端處的前 20 個核苷酸。在某些實施例中，間隔區中之修飾不會幹擾編輯效率，其可以使用此項技術中已知或本文所述之技術來評估。在某些實施例中，間隔區中之修飾可以提高編輯效率且減少脫靶效應。在某些實施例中，間隔區包括 1、2、3、4、5、6、7、8、9 或 10 個修飾。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括 1、2、3、4、5、6、7、8、9 或 10 個修飾。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括 1、2、3、4、5 或 6 個修飾。在某些實施例中，間隔區在其 5' 端處的前 3 個核苷酸內包括 1、2、3、4、5、6、7、8、9 或 10 個修飾。在某些實施例中，間隔區在其 5' 端處的前 3 個核苷酸內包括 1、2、3、4、5 或 6 個修飾。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括至少一個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括 1、2、3、4 或 5 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括 1 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括 2 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括 3 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括 4 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 5 個核苷酸內包括 5 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 3 個核苷酸內包括至少一個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 3 個核苷酸內包括 1、2 或 3 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 3 個核苷酸內包括 1 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 3 個核苷酸內包括 2 個化學修飾之核苷酸。在某些實施例中，間隔區在其 5' 端處的前 3 個核苷酸內包括 3 個化學修飾之核苷酸。在某些實施例中，化學修飾之核苷酸可包括一或多個修飾、兩個或更多個修飾或三個或更多個修飾。在某些實施例中，化學修飾之核苷酸可包括一個修飾，例如，糖修飾或磷酸主鏈修飾，如下文所述。在某些實施例中，化學修飾之核苷酸可包括兩個修飾，例如，糖修飾及磷酸主鏈修飾，如下文所述。在某些實施例中，化學修飾之核苷酸可包括三個修飾，例如，兩個糖修飾及一個磷酸主鏈修飾，如下文所述。In certain embodiments, the gRNA molecules disclosed herein include one or more modifications in the spacer region of the gRNA molecule. In certain embodiments, the spacer region of the gRNA molecules disclosed herein is complementary to the sequence of the target nucleic acid, for example, at least about 80%, about 85%, about 90%, 95%, about 98%, about 99%, or about 100% complementary. In certain embodiments, the spacer region includes the first 30 nucleotides present at the 5' end of the gRNA molecule, for example, the first 29 nucleotides, the first 28 nucleotides, the first 27 nucleotides, the first 26 nucleotides, the first 25 nucleotides, the first 24 nucleotides, the first 23 nucleotides, the first 22 nucleotides, the first 21 nucleotides, the first 20 nucleotides, the first 19 nucleotides, the first 18 nucleotides, the first 17 nucleotides, the first 16 nucleotides, or the first 15 nucleotides. In some embodiments, the spacer region includes the first 20 nucleotides present at the 5' end of the gRNA molecule. In some embodiments, modifications in the spacer region do not interfere with editing efficiency, which can be assessed using techniques known in the art or described herein. In some embodiments, modifications in the spacer region can increase editing efficiency and reduce off-target effects. In some embodiments, the spacer region includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications. In some embodiments, the spacer region includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications within the first 5 nucleotides at its 5' end. In some embodiments, the spacer region includes 1, 2, 3, 4, 5 or 6 modifications within the first 5 nucleotides at its 5' end. In certain embodiments, the spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer comprises 1, 2, 3, 4, 5 or 6 modifications within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer comprises at least one chemically modified nucleotide within the first 5 nucleotides at its 5' end. In certain embodiments, the spacer comprises 1, 2, 3, 4 or 5 chemically modified nucleotides within the first 5 nucleotides at its 5' end. In certain embodiments, the spacer comprises 1 chemically modified nucleotide within the first 5 nucleotides at its 5' end. In certain embodiments, the spacer comprises 2 chemically modified nucleotides within the first 5 nucleotides at its 5' end. In certain embodiments, the spacer comprises 3 chemically modified nucleotides within the first 5 nucleotides at its 5' end. In certain embodiments, the spacer comprises 4 chemically modified nucleotides within the first 5 nucleotides at its 5' end. In certain embodiments, the spacer comprises 5 chemically modified nucleotides within the first 5 nucleotides at its 5' end. In certain embodiments, the spacer comprises at least one chemically modified nucleotide within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer comprises 1, 2 or 3 chemically modified nucleotides within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer comprises 1 chemically modified nucleotide within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer comprises 2 chemically modified nucleotides within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer includes 3 chemically modified nucleotides within the first 3 nucleotides at its 5' end. In certain embodiments, the chemically modified nucleotides may include one or more modifications, two or more modifications, or three or more modifications. In certain embodiments, the chemically modified nucleotides may include one modification,for example , a sugar modification or a phosphate backbone modification, as described below. In certain embodiments, the chemically modified nucleotides may include two modifications,for example , a sugar modification and a phosphate backbone modification, as described below. In certain embodiments, the chemically modified nucleotides may include three modifications,for example , two sugar modifications and one phosphate backbone modification, as described below.

在某些實施例中，本揭露之 gRNA 分子在其 5' 端處包括一或多個修飾。在某些實施例中，如本文所用之關於修飾的術語「5' 端」係指 gRNA 分子之 5' 端處的前 5 個核苷酸。例如但不作為限制，存在於本揭露之 gRNA 分子之 5' 端處的一或多個核苷酸被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 5' 端處的第一個核苷酸被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 5' 端處的第二個核苷酸被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 5' 端處的第三個核苷酸被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 5' 端處的第四個核苷酸被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 5' 端處的第五個核苷酸被修飾。在某些實施例中，本揭露之 gRNA 分子在其 5' 端處的前 5 個核苷酸內包括 1、2、3、4、5、6、7、8、9 或 10 個修飾。In certain embodiments, the gRNA molecules of the present disclosure include one or more modifications at their 5' end. In certain embodiments, the term "5' end" as used herein with respect to modifications refers to the first 5 nucleotides at the 5' end of the gRNA molecule. For example, but not by way of limitation, one or more nucleotides present at the 5' end of the gRNA molecule of the present disclosure are modified. In certain embodiments, the first nucleotide present at the 5' end of the gRNA molecule of the present disclosure is modified. In certain embodiments, the second nucleotide present at the 5' end of the gRNA molecule of the present disclosure is modified. In certain embodiments, the third nucleotide present at the 5' end of the gRNA molecule of the present disclosure is modified. In certain embodiments, the fourth nucleotide present at the 5' end of the gRNA molecule of the present disclosure is modified. In certain embodiments, the fifth nucleotide present at the 5' end of the gRNA molecule of the present disclosure is modified. In certain embodiments, the gRNA molecule of the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications within the first 5 nucleotides at its 5' end.

在某些實施例中，本揭露之 gRNA 分子包括兩個連續核苷酸，例如，在 gRNA 分子之 5' 端的前 5 個核苷酸內的兩個連續核苷酸處的修飾。例如但不作為限制，gRNA 分子之第一個與第二個核苷酸被修飾。在某些實施例中，gRNA 分子之第一個與第三個核苷酸被修飾。在某些實施例中，gRNA 分子之 5' 端處的第二個與第三個核苷酸被修飾。在某些實施例中，gRNA 分子之 5' 端處的第三個與第四個核苷酸被修飾。在某些實施例中，gRNA 分子之 5' 端處的第四個與第五個核苷酸被修飾。In some embodiments, the gRNA molecules disclosed herein include two consecutive nucleotides, for example, modifications at two consecutive nucleotides within the first 5 nucleotides of the 5' end of the gRNA molecule. For example, but not by way of limitation, the first and second nucleotides of the gRNA molecule are modified. In some embodiments, the first and third nucleotides of the gRNA molecule are modified. In some embodiments, the second and third nucleotides at the 5' end of the gRNA molecule are modified. In some embodiments, the third and fourth nucleotides at the 5' end of the gRNA molecule are modified. In some embodiments, the fourth and fifth nucleotides at the 5' end of the gRNA molecule are modified.

在某些實施例中，本揭露之 gRNA 分子包括在三個連續核苷酸，例如， gRNA 分子之 5' 端的前 5 個核苷酸內的三個連續核苷酸處的修飾。例如但不作為限制，gRNA 分子之 5' 端處的第一個、第二個與第三個核苷酸被修飾。在某些實施例中，gRNA 分子之 5' 端處的第二個、第三個與第四個核苷酸被修飾。在某些實施例中，gRNA 分子之 5' 端處的第三個、第四個與第五個核苷酸被修飾。In certain embodiments, the gRNA molecules disclosed herein include modifications at three consecutive nucleotides, for example, three consecutive nucleotides within the first 5 nucleotides of the 5' end of the gRNA molecule. For example, but not by way of limitation, the first, second, and third nucleotides at the 5' end of the gRNA molecule are modified. In certain embodiments, the second, third, and fourth nucleotides at the 5' end of the gRNA molecule are modified. In certain embodiments, the third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are modified.

在某些實施例中，本揭露之 gRNA 分子包括在四個連續核苷酸，例如，gRNA 分子之 5' 端的前 5 個核苷酸內的四個連續核苷酸處的修飾。例如但不作為限制，gRNA 分子之 5' 端處的第一個、第二個、第三個與第四個核苷酸被修飾。在某些實施例中，gRNA 分子之 5' 端處的第二個、第三個、第四個與第五個核苷酸被修飾。In certain embodiments, the gRNA molecules disclosed herein include modifications at four consecutive nucleotides, for example, at four consecutive nucleotides within the first 5 nucleotides at the 5' end of the gRNA molecule. For example, but not by way of limitation, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are modified. In certain embodiments, the second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are modified.

在某些實施例中，本揭露之 gRNA 分子包括在五個連續核苷酸，例如，gRNA 分子之 5' 端的前 5 個核苷酸內的五個連續核苷酸處的修飾。例如但不作為限制，gRNA 分子之 5' 端處的第一個、第二個、第三個、第四個與第五個核苷酸被修飾。In certain embodiments, the gRNA molecule disclosed herein includes modifications at five consecutive nucleotides, for example, five consecutive nucleotides within the first 5 nucleotides at the 5' end of the gRNA molecule. For example, but not by way of limitation, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are modified.

在某些實施例中，本揭露之 gRNA 分子在其 3' 端處包括一或多個修飾。在某些實施例中，如本文所用之關於修飾的術語「3' 端」係指 gRNA 分子之 3' 端處的最後 5 個核苷酸 (第 n 個、第 n-1 個、第 n-2 個、第 n-3 個及第 n-4 個核苷酸)。例如但不作為限制，存在於本揭露之 gRNA 分子之 3' 端處的一或多個核苷酸被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 3' 端處的最後一個核苷酸 (第 n 個) 被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 3' 端處的倒數第二個核苷酸 (第 n-1 個) 被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 3' 端處的倒數第三個核苷酸 (第 n-2 個) 被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 3' 端處的倒數第四個核苷酸 (第 n-3 個) 被修飾。在某些實施例中，存在於本揭露之 gRNA 分子之 3' 端處的倒數第五個核苷酸 (第 n-4 個) 被修飾。在某些實施例中，本揭露之 gRNA 分子在其 3' 端處的最後 5 個核苷酸內包括 1、2、3、4、5、6、7、8、9 或 10 個修飾。In certain embodiments, the gRNA molecules disclosed herein include one or more modifications at their 3' end. In certain embodiments, the term "3' end" as used herein with respect to modifications refers to the last 5 nucleotides (nth, n-1st, n-2nd, n-3rd, and n-4th nucleotides) at the 3' end of the gRNA molecule. For example, but not by way of limitation, one or more nucleotides present at the 3' end of the gRNA molecule disclosed herein are modified. In certain embodiments, the last nucleotide (nth) present at the 3' end of the gRNA molecule disclosed herein is modified. In certain embodiments, the penultimate nucleotide (n-1st) present at the 3' end of the gRNA molecule disclosed herein is modified. In certain embodiments, the third to last nucleotide (n-2) present at the 3' end of the gRNA molecule of the present disclosure is modified. In certain embodiments, the fourth to last nucleotide (n-3) present at the 3' end of the gRNA molecule of the present disclosure is modified. In certain embodiments, the fifth to last nucleotide (n-4) present at the 3' end of the gRNA molecule of the present disclosure is modified. In certain embodiments, the gRNA molecule of the present disclosure includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications within the last 5 nucleotides at its 3' end.

在某些實施例中，本揭露之 gRNA 分子包括在兩個連續核苷酸，例如， gRNA 分子之 3' 端的 5 個核苷酸內的兩個連續核苷酸處的修飾。例如但不作為限制，gRNA 分子之 3' 端的第 n 個與第 n-1 個核苷酸被修飾。在某些實施例中，gRNA 分子之 3' 端的第 n 個與第 n-2 個核苷酸被修飾。在某些實施例中，gRNA 分子之 3' 端的第 n-1 個與第 n-2 個核苷酸被修飾。在某些實施例中，gRNA 分子之 3' 端的第 n-2 個與第 n-3 個核苷酸被修飾。在某些實施例中，gRNA 分子之 3' 端的第 n-3 個與第 n-4 個核苷酸被修飾。在某些實施例中，gRNA 分子之 3' 端的第 n-4 個與第 n-5 個核苷酸被修飾。In certain embodiments, the gRNA molecules disclosed herein include modifications at two consecutive nucleotides, for example, two consecutive nucleotides within 5 nucleotides of the 3' end of the gRNA molecule. For example, but not by way of limitation, the nth and n-1th nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the nth and n-2th nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-1th and n-2th nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-2th and n-3th nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-3th and n-4th nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-4th and n-5th nucleotides at the 3' end of the gRNA molecule are modified.

在某些實施例中，本揭露之 gRNA 分子包括在三個連續核苷酸，例如，gRNA 分子之 3' 端的最後 5 個核苷酸內的三個連續核苷酸處的修飾。例如但不作為限制，gRNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸被修飾。在某些實施例中，gRNA 分子之 3' 端的第 n-1 個、第 n-2 個與第 n-3 個核苷酸被修飾。在某些實施例中，gRNA 分子之 3' 端的第 n-2 個、第 n-3 個與第 n-4 個核苷酸被修飾。In certain embodiments, the gRNA molecules disclosed herein include modifications at three consecutive nucleotides, for example, three consecutive nucleotides within the last 5 nucleotides at the 3' end of the gRNA molecule. For example, but not by way of limitation, the nth, n-1th, and n-2th nucleotides of the gRNA molecule are modified. In certain embodiments, the n-1th, n-2th, and n-3th nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-2th, n-3th, and n-4th nucleotides at the 3' end of the gRNA molecule are modified.

在某些實施例中，本揭露之 gRNA 分子包括在四個連續核苷酸，例如，gRNA 分子之 3' 端的 5 個核苷酸內的四個連續核苷酸處的修飾。例如但不作為限制，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸被修飾。在某些實施例中，gRNA 分子之第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸被修飾。In certain embodiments, the gRNA molecules disclosed herein include modifications at four consecutive nucleotides, for example, four consecutive nucleotides within 5 nucleotides of the 3' end of the gRNA molecule. For example, but not by way of limitation, the nth, n-1th, n-2th, and n-3th nucleotides of the gRNA molecule are modified. In certain embodiments, the n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule are modified.

在某些實施例中，本揭露之 gRNA 分子包括在五個連續核苷酸，例如， gRNA 分子之 3' 端的 5 個核苷酸內的五個連續核苷酸處的修飾。例如但不作為限制，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸被修飾。In certain embodiments, the gRNA molecule disclosed herein includes modifications at five consecutive nucleotides, for example, five consecutive nucleotides within 5 nucleotides of the 3' end of the gRNA molecule. For example, but not by way of limitation, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule are modified.

在某些實施例中，本揭露之 gRNA 分子包括在其 5' 端處的一或多個修飾以及在其 3' 端處的一或多個修飾。在某些實施例中，本揭露之 gRNA 分子包括在其 5' 端處的兩個或更多個修飾以及在其 3' 端處的兩個或更多個修飾。在某些實施例中，本揭露之 gRNA 分子包括在其 5' 端處的三個或更多個修飾以及在其 3' 端處的三個或更多個修飾。在某些實施例中，本揭露之 gRNA 分子包括在其 5' 端處的四個或更多個修飾以及在其 3' 端處的四個或更多個修飾。在某些實施例中，本揭露之 gRNA 分子包括在其 5' 端處的五個或更多個修飾以及在其 3' 端處的五個或更多個修飾。In certain embodiments, the gRNA molecules of the present disclosure include one or more modifications at its 5' end and one or more modifications at its 3' end. In certain embodiments, the gRNA molecules of the present disclosure include two or more modifications at its 5' end and two or more modifications at its 3' end. In certain embodiments, the gRNA molecules of the present disclosure include three or more modifications at its 5' end and three or more modifications at its 3' end. In certain embodiments, the gRNA molecules of the present disclosure include four or more modifications at its 5' end and four or more modifications at its 3' end. In certain embodiments, the gRNA molecules of the present disclosure include five or more modifications at its 5' end and five or more modifications at its 3' end.

本文揭示了可包括於本揭露之 gRNA 分子中的修飾之非限制性實例。在某些實施例中，本揭露之 gRNA 可包括以下修飾中的一或多個：(i) 磷酸主鏈修飾及 (ii) 糖修飾。 磷酸主鏈修飾Disclosed herein are non-limiting examples of modifications that may be included in the gRNA molecules of the present disclosure. In certain embodiments, the gRNA of the present disclosure may include one or more of the following modifications: (i) phosphate backbone modifications and (ii) sugar modifications.Phosphate backbone modifications

在某些實施例中，gRNA 分子內存在之核苷酸的磷酸基團可被修飾。例如但不作為限制，核苷酸之磷酸基團可以藉由用不同的取代基替換磷酸二酯鍵結中的一或多個氧 (例如橋接氧或非橋接氧) 來修飾。取代基之非限制性實例包括硫 (S)、氮 (N)、氫 (H) 及碳 (C)。在某些實施例中，磷酸二酯鍵結中之一或多個氧用 S 取代。In certain embodiments, the phosphate groups of the nucleotides present in the gRNA molecule can be modified. For example, but not by way of limitation, the phosphate groups of the nucleotides can be modified by replacing one or more oxygens in the phosphodiester bond with different substituents(e.g., bridging oxygens or non-bridging oxygens). Non-limiting examples of substituents include sulfur (S), nitrogen (N), hydrogen (H), and carbon (C). In certain embodiments, one or more oxygens in the phosphodiester bond are replaced with S.

在某些實施例中，gRNA 分子可用一或多個硫代磷酸酯 (PS) 鍵結進行修飾。在某些實施例中，PS 鍵結或鍵係指其中硫取代核苷酸間磷酸二酯鍵結中之一個非橋接磷酸氧之鍵。在某些實施例中，「*」在本文中用於指示藉由 PS 鍵結連接至相鄰的 3' 核苷酸之核苷酸。在某些實施例中，未經修飾的磷酸二酯鍵結之磷為非掌性的且用硫替換一個非橋接磷酸氧賦予磷掌性。In certain embodiments, the gRNA molecule can be modified with one or more phosphorothioate (PS) bonds. In certain embodiments, a PS bond or bonds refers to a bond in which sulfur replaces one of the non-bridging phosphate oxygens in an internucleotide phosphodiester bond. In certain embodiments, "*" is used herein to indicate a nucleotide that is linked to the adjacent 3' nucleotide via a PS bond. In certain embodiments, the phosphorus of the unmodified phosphodiester bond is non-chiral and replacing one of the non-bridging phosphate oxygens with sulfur imparts chirality to the phosphorus.

在某些實施例中，gRNA 分子可用一或多個二硫代磷酸酯 (PS2) 鍵結進行修飾。在某些實施例中，PS2 鍵結或鍵係指其中核苷酸間磷酸二酯鍵結中的兩個非橋接氧被硫替換的鍵。在某些實施例中，「**」在本文中用於指示藉由PS2 鍵結連接至相鄰的 3' 核苷酸之核苷酸。類似於天然存在之磷酸二酯主鏈鍵結，PS2 鍵結在磷處為非掌性的，導致 gRNA 分子在 PS2 鍵結處不為非鏡像異構物。在某些實施例中，二硫代磷酸酯鍵結對核酸酶降解具有抗性，且例如與未經修飾的 gRNA 相比，本揭露之 gRNA 中一或多個二硫代磷酸酯鍵結之存在可以增加 gRNA 之穩定性。 糖修飾In certain embodiments, the gRNA molecule may be modified with one or more phosphorodithioate (PS2) linkages. In certain embodiments, a PS2 linkage or linkage refers to a linkage in which the two non-bridging oxygens in an internucleotide phosphodiester linkage are replaced by sulfur. In certain embodiments, "**" is used herein to indicate a nucleotide that is linked to the adjacent 3' nucleotide via a PS2 linkage. Similar to naturally occurring phosphodiester backbone linkages, the PS2 linkage is non-chiral at the phosphorus, resulting in the gRNA molecule not being a non-mirror isomer at the PS2 linkage. In certain embodiments, the phosphorodithioate bonds are resistant to nuclease degradation, and the presence of one or more phosphorodithioate bonds in the gRNA of the present disclosure can increase the stability of the gRNA, for example, compared to an unmodified gRNA.Sugar Modifications

在某些實施例中，本揭露之 gRNA 中存在的核苷酸之糖基團可以被修飾。例如但不作為限制，本揭露之 gRNA 之核苷酸可以包括對其糖基團 (例如，核糖) 之一或多個修飾。In certain embodiments, the sugar groups of the nucleotides present in the gRNA disclosed herein may be modified. For example, but not by way of limitation, the nucleotides of the gRNA disclosed herein may include one or more modifications to their sugar groups(e.g., ribose).

在某些實施例中，糖基團可以在 2' 羥基 (OH) 處被修飾。在某些實施例中，2' 羥基可以經不同的取代基替換。取代基之非限制性實例包括氫 (H)、鹵素、烷基或烷氧基 (OR，其中 R 可以為烷基、環烷基或烷氧基)。In some embodiments, the sugar group can be modified at the 2' hydroxyl (OH). In some embodiments, the 2' hydroxyl can be replaced by various substituents. Non-limiting examples of substituents include hydrogen (H), halogen, alkyl or alkoxy (OR, where R can be alkyl, cycloalkyl or alkoxy).

在某些實施例中，2' 羥基經烷氧基取代。在某些實施例中，2' 羥基經甲氧基取代。在某些實施例中，「m」在本文中用於指示經 2'-O-甲基修飾的核苷酸 (即，經 2'-O-甲基修飾的核苷酸)。In certain embodiments, the 2'hydroxyl group is substituted with an alkoxy group. In certain embodiments, the 2'hydroxyl group is substituted with a methoxy group. In certain embodiments, "m" is used herein to indicate a 2'-O-methyl modified nucleotide(i.e., a 2'-O-methyl modified nucleotide).

在某些實施例中，2' 羥基之氫 (H) 經甲氧基乙基取代。在某些實施例中，「M」在本文中用於指示經 2'-O-(2-甲氧基乙基) 修飾的核苷酸 (即，經 2'--(2-甲氧基乙基) 修飾的核苷酸)。In certain embodiments, the hydrogen (H) of the 2' hydroxyl group is substituted with a methoxyethyl group. In certain embodiments, "M" is used herein to indicate a 2'-O-(2-methoxyethyl)-modified nucleotide(i.e., a 2'--(2-methoxyethyl)-modified nucleotide).

在某些實施例中，2' 羥基可以經鹵素取代。鹵素之非限制性實例包括氟 (F)、氯 (Cl)、溴化物 (Br) 及碘 (I)。在某些實施例中，2' 羥基經氟替換。在某些實施例中，「f」在本文中用於指示經 2'-氟修飾的核苷酸 (即，經 2'-氟修飾的核苷酸)。In certain embodiments, the 2' hydroxyl group may be substituted with a halogen. Non-limiting examples of halogens include fluorine (F), chlorine (Cl), bromide (Br), and iodine (I). In certain embodiments, the 2' hydroxyl group is substituted with a fluorine. In certain embodiments, "f" is used herein to indicate a 2'-fluorine-modified nucleotide(i.e., a 2'-fluorine-modified nucleotide).

在某些實施例中，2' 羥基可以經氫 (H) 替換以產生去氧核糖。例如但不作為限制，本揭露之 gRNA 中存在的核苷酸可以具有去氧核糖。在某些實施例中，「d」在本文中用於指示具有去氧核糖之核苷酸。In certain embodiments, the 2' hydroxyl group can be replaced by hydrogen (H) to generate a deoxyribose. For example, but not by way of limitation, the nucleotides present in the gRNA of the present disclosure can have a deoxyribose. In certain embodiments, "d" is used herein to indicate a nucleotide having a deoxyribose.

在某些實施例中，2' 羥基之修飾可包括「鎖核酸」(LNA)，其中 2' 羥基連接至相同核糖之 4' 碳。在某些實施例中，2' 羥基藉由橋 (例如，亞烷基 (例如，亞甲基)、醚或胺基橋) 連接至 4' 碳。在某些實施例中，「LNA」在本文中用於指示作為 LNA 之核苷酸。 例示性經修飾之 gRNAIn certain embodiments, modification of the 2' hydroxyl group may include a "locked nucleic acid" (LNA), wherein the 2' hydroxyl group is linked to the 4' carbon of the same ribose. In certain embodiments, the 2' hydroxyl group is linked to the 4' carbon via a bridge(e.g., an alkylene(e.g., a methylene), ether, or amine bridge). In certain embodiments, "LNA" is used herein to indicate a nucleotide that is an LNA.Exemplary modified gRNAs

在某些實施例中，本揭露之 gRNA 可具有至少一個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少兩個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少三個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少四個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少五個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少六個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少七個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少八個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少九個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有至少十個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有約 1 至約 10 個磷酸主鏈修飾，例如約 1 至約 9、約 1 至約 8、約 1 至約 7、約 1 至約 6、約 1 至約 5、約 1 至約 4、約 1 至約 3、約 1 至約 2、約 2 至約 10、約 3 至約 10、約 4 至約 10、約 5 至約 10、約 6 至約 10、約 7 至約 10、約 8 至約 10、約 9 至約 10、約 2 至約 8、約 2 至約 6、約 3 至約 8、約 3 至約 6 或 3 至 5 個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有約 2 至約 10 個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有約 2 至約 9 個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有約 3 至約 8 個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。在某些實施例中，gRNA 可具有約 3 至約 5 個磷酸主鏈修飾，例如，二硫代磷酸酯鍵結。In certain embodiments, the gRNA disclosed herein may have at least one phosphate backbone modification, such as a phosphorodithioate linkage. In certain embodiments, the gRNA may have at least two phosphate backbone modifications, such as a phosphorodithioate linkage. In certain embodiments, the gRNA may have at least three phosphate backbone modifications, such as a phosphorodithioate linkage. In certain embodiments, the gRNA may have at least four phosphate backbone modifications, such as a phosphorodithioate linkage. In certain embodiments, the gRNA may have at least five phosphate backbone modifications, such as a phosphorodithioate linkage. In certain embodiments, the gRNA may have at least six phosphate backbone modifications, such as a phosphorodithioate linkage. In certain embodiments, the gRNAmay have at least seven phosphate backbone modifications, e.g., phosphorodithioate linkages. In certain embodiments, the gRNAmay have at least eight phosphate backbone modifications, e.g., phosphorodithioate linkages. In certain embodiments, the gRNAmay have at least nine phosphate backbone modifications, e.g., phosphorodithioate linkages. In certain embodiments, the gRNAmay have at least ten phosphate backbone modifications, e.g., phosphorodithioate linkages. In certain embodiments, the gRNA may have about 1 to about 10 phosphate backbone modifications, such as about1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 9 to about 10, about 2 to about 8, about 2 to about 6, about 3 to about 8, about 3 to about 6, or 3 to 5In some embodiments, the gRNA may have about 2 to about 10phosphate backbone modifications, such as phosphorodithioate linkages. In some embodiments, the gRNA may have about 2 to about 9phosphate backbone modifications, such as phosphorodithioate linkages. In some embodiments, the gRNA may have about 3 to about 8phosphate backbone modifications, such as phosphorodithioate linkages. In some embodiments, the gRNA may have about 3 to about 5phosphate backbone modifications, such as phosphorodithioate linkages.

在某些實施例中，本揭露之 gRNA 可包括至少一個二硫代磷酸酯鍵結及至少一個硫代磷酸酯鍵結，例如，如表 10 中所示。例如但不作為限制，本揭露之 gRNA 可包括至少兩個二硫代磷酸酯鍵結及至少一個硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 可包括至少三個二硫代磷酸酯鍵結及至少一個硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 可包括至少四個二硫代磷酸酯鍵結及至少一個硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 可包括至少兩個二硫代磷酸酯鍵結及至少兩個硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 可包括至少兩個二硫代磷酸酯鍵結及至少三個硫代磷酸酯鍵結。In certain embodiments, the gRNA disclosed herein may include at least one phosphorodithioate bond and at least one phosphorothioate bond, for example, as shown in Table10. For example, but not by way of limitation, the gRNA disclosed herein may include at least two phosphorodithioate bonds and at least one phosphorothioate bond. In certain embodiments, the gRNA disclosed herein may include at least three phosphorodithioate bonds and at least one phosphorothioate bond. In certain embodiments, the gRNA disclosed herein may include at least four phosphorodithioate bonds and at least one phosphorothioate bond. In certain embodiments, the gRNA disclosed herein may include at least two phosphorodithioate bonds and at least two phosphorothioate bonds. In certain embodiments, the gRNA disclosed herein may include at least two phosphorodithioate bonds and at least three phosphorothioate bonds.

在某些實施例中，gRNA 可具有至少一個糖修飾。在某些實施例中，gRNA 可具有至少兩個糖修飾。在某些實施例中，gRNA 可具有至少三個糖修飾。在某些實施例中，gRNA 可具有至少四個糖修飾。在某些實施例中，gRNA 可具有至少五個糖修飾。在某些實施例中，gRNA 可具有至少六個糖修飾。在某些實施例中，gRNA 可具有至少七個糖修飾。在某些實施例中，gRNA 可具有至少八個糖修飾。在某些實施例中，gRNA 可具有至少九個糖修飾。在某些實施例中，gRNA 可具有至少十個糖修飾。在某些實施例中，gRNA 可具有約 1 至約 10 個糖修飾，例如約 1 至約 9、約 1 至約 8、約 1 至約 7、約 1 至約 6、約 1 至約 5、約 1 至約 4、約 1 至約 3、約 1 至約 2、約 2 至約 10、約 3 至約 10、約 4 至約 10、約 5 至約 10、約 6 至約 10、約 7 至約 10、約 8 至約 10、約 9 至約 10、約 2 至約 8、約 2 至約 6、約 3 至約 8 或約 3 至約 6 個糖修飾。在某些實施例中，gRNA 可具有約 2 至約 10 個糖修飾。在某些實施例中，gRNA 可具有約 3 至約 8 個糖修飾。在某些實施例中，本揭露之 gRNA 分子可具有約 3 至約 6 個糖修飾。In certain embodiments, the gRNA may have at least one sugar modification. In certain embodiments, the gRNA may have at least two sugar modifications. In certain embodiments, the gRNA may have at least three sugar modifications. In certain embodiments, the gRNA may have at least four sugar modifications. In certain embodiments, the gRNA may have at least five sugar modifications. In certain embodiments, the gRNA may have at least six sugar modifications. In certain embodiments, the gRNA may have at least seven sugar modifications. In certain embodiments, the gRNA may have at least eight sugar modifications. In certain embodiments, the gRNA may have at least nine sugar modifications. In certain embodiments, the gRNA may have at least ten sugar modifications. In certain embodiments, the gRNA can have about 1 to about 10 sugar modifications, such as about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 9 to about 10, about 2 to about 8, about 2 to about 6, about 3 to about 8, or about 3 to about 6 sugar modifications. In certain embodiments, the gRNA may have about 2 to about 10 sugar modifications. In certain embodiments, the gRNA may have about 3 to about 8 sugar modifications. In certain embodiments, the gRNA molecules of the present disclosure may have about 3 to about 6 sugar modifications.

在某些實施例中，gRNA 可具有至少一個磷酸主鏈修飾 (例如，至少兩個、至少三個、至少四個、至少五個、至少六個、至少七個、至少八個、至少九個或至少十個磷酸主鏈修飾) 以及至少一個糖修飾 (例如，至少兩個、至少三個、至少四個、至少五個、至少六個、至少七個、至少八個、至少九個或至少十個糖修飾)。In certain embodiments, the gRNA can have at least one phosphate backbone modification(e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten phosphate backbone modifications) and at least one sugar modification(e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten sugar modifications).

在某些實施例中，gRNA 可具有至少兩個磷酸主鏈修飾及至少兩個糖修飾。在某些實施例中，gRNA 可具有至少三個磷酸主鏈修飾及至少三個糖修飾。在某些實施例中，gRNA 可具有至少四個磷酸主鏈修飾及至少四個糖修飾。在某些實施例中，gRNA 可具有至少五個磷酸主鏈修飾及至少五個糖修飾。在某些實施例中，gRNA 可具有至少五個磷酸主鏈修飾及至少六個糖修飾。在某些實施例中，gRNA 可具有至少六個磷酸主鏈修飾及至少六個糖修飾。在某些實施例中，gRNA 可具有至少七個磷酸主鏈修飾及至少七個糖修飾。在某些實施例中，gRNA 可具有至少八個磷酸主鏈修飾及至少八個糖修飾。在某些實施例中，gRNA 可具有至少八個磷酸主鏈修飾及至少九個糖修飾。在某些實施例中，gRNA 可具有至少九個磷酸主鏈修飾及至少九個糖修飾。在某些實施例中，gRNA 可具有至少十個磷酸主鏈修飾及至少十個糖修飾。In certain embodiments, the gRNA may have at least two phosphate backbone modifications and at least two sugar modifications. In certain embodiments, the gRNA may have at least three phosphate backbone modifications and at least three sugar modifications. In certain embodiments, the gRNA may have at least four phosphate backbone modifications and at least four sugar modifications. In certain embodiments, the gRNA may have at least five phosphate backbone modifications and at least five sugar modifications. In certain embodiments, the gRNA may have at least five phosphate backbone modifications and at least six sugar modifications. In certain embodiments, the gRNA may have at least six phosphate backbone modifications and at least six sugar modifications. In certain embodiments, the gRNA may have at least seven phosphate backbone modifications and at least seven sugar modifications. In certain embodiments, the gRNA may have at least eight phosphate backbone modifications and at least eight sugar modifications. In certain embodiments, the gRNA may have at least eight phosphate backbone modifications and at least nine sugar modifications. In certain embodiments, the gRNA may have at least nine phosphate backbone modifications and at least nine sugar modifications. In certain embodiments, the gRNA may have at least ten phosphate backbone modifications and at least ten sugar modifications.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的至少一個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第二個與第三個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第四個與第五個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules of the present disclosure include at least one phosphorodithioate bond at the 5' end of the gRNA molecule. In certain embodiments, the gRNA molecules of the present disclosure include a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the gRNA molecules of the present disclosure include a phosphorodithioate bond between the second and third nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the gRNA molecules of the present disclosure include a phosphorodithioate bond between the third and fourth nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the gRNA molecules of the present disclosure include a phosphorodithioate bond between the fourth and fifth nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 3' 端處的至少一個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 3' 端 (第「n」個) 核苷酸與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 3' 端處的第 n-1 個與第 n-2 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 3' 端處的第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 3' 端處的第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules of the present disclosure include at least one phosphorodithioate bond at the 3' end of the gRNA molecule. In certain embodiments, the gRNA molecules of the present disclosure include a phosphorodithioate bond between the ("n"th) nucleotide and the n-1th nucleotide at the 3' end of the gRNA molecule. In certain embodiments, the gRNA molecules of the present disclosure include a phosphorodithioate bond between the n-1th and n-2th nucleotides at the 3' end of the gRNA molecule. In certain embodiments, the gRNA molecules of the present disclosure include a phosphorodithioate bond between the n-2th and n-3th nucleotides at the 3' end of the gRNA molecule. In certain embodiments, the gRNA molecules disclosed herein include a phosphorodithioate bond between the n-3rd and n-4th nucleotides at the 3' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結，以及在 gRNA 分子之第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule, and a phosphorodithioate bond between the nth and n-1th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸以及第二個與第三個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the first and second nucleotides and the second and third nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the first and second nucleotides and the third and fourth nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第二個與第三個核苷酸以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the second and third nucleotides and the third and fourth nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、第三個與第四個核苷酸以及第四個與第五個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the first and second nucleotides, the second and third nucleotides, the third and fourth nucleotides, and the fourth and fifth nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在第一個與第二個核苷酸之間的硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include a phosphorothioate bond between the first and second nucleotides.

在某些實施例中，本揭露之 gRNA 分子包括在第三個與第四個核苷酸之間的硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include a phosphorothioate bond between the third and fourth nucleotides.

在某些實施例中，本揭露之 gRNA分子包括在第一個與第二個核苷酸、第二個與第三個核苷酸以及第三個與第四個核苷酸之間的硫代磷酸酯鍵結。In certain embodiments, the gRNAmolecules disclosed herein include phosphorothioate bonds between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides.

在某些實施例中，本揭露之 gRNA 分子包括在第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之第 n-1 個與第 n-2 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the n-1 and n-2 nucleotides and the n-2 and n-3 nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個、第 n-1 個與第 n-2 個核苷酸、第 n-2 個與第 n-3 個以及第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the nth and n-1th, n-1th and n-2th nucleotides, n-2th and n-3th, and n-3th and n-4th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在 gRNA 分子之第 n-1 個與第 n-2 個核苷酸、第 n-2 個與第 n-3 個、第 n-3 個與第 n-4 個以及第 n-4 個與第 n-5 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorodithioate bonds between the n-1st and n-2nd nucleotides, the n-2nd and n-3rd, the n-3rd and n-4th, and the n-4th and n-5th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 分子包括在第 n 個與第 n-1 個核苷酸之間的硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include a phosphorothioate bond between the nth and n-1th nucleotides.

在某些實施例中，本揭露之 gRNA 分子包括在第 n-1 個與第 n-2 個核苷酸之間的硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include a phosphorothioate bond between the n-1 and n-2 nucleotides.

在某些實施例中，本揭露之 gRNA 分子包括在第 n-1 個與第 n-2 個核苷酸之間的硫代磷酸酯鍵結以及在第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 進一步包括在第一個與第二個核苷酸、第二個與第三個核苷酸以及第三個與第四個核苷酸之間的硫代磷酸酯鍵結。In certain embodiments, the gRNA molecule disclosed herein comprises a phosphorothioate bond between the n-1st and n-2nd nucleotides and a phosphorodithioate bond between the nth and n-1st nucleotides. In certain embodiments, the gRNA further comprises a phosphorothioate bond between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides.

在某些實施例中，本揭露之 gRNA 分子包括在第三個與第四個核苷酸之間的硫代磷酸酯鍵結以及在第一個與第二個核苷酸以及第二個與第三個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 進一步包括在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個核苷酸之間的二硫代磷酸酯鍵結In certain embodiments, the gRNA molecule disclosed herein comprises a phosphorothioate bond between the third and fourth nucleotides and a phosphorodithioate bond between the first and second nucleotides and between the second and third nucleotides. In certain embodiments, the gRNA further comprises a phosphorodithioate bond between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides

在某些實施例中，本揭露之 gRNA 分子包括在第一個與第二個核苷酸、第二個與第三個核苷酸以及第三個與第四個核苷酸之間的硫代磷酸酯鍵結。在某些實施例中，gRNA 進一步包括在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA molecules disclosed herein include phosphorothioate bonds between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides. In certain embodiments, the gRNA further includes phosphorodithioate bonds between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides.

在某些實施例中，本揭露之 gRNA 分子在其 5' 端處，例如在 5' 端處的前 5 個核苷酸內包括經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及鎖核酸 (LNA) 中之一或多個。例如但不作為限制，gRNA 分子之 5' 端處的第一個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或鎖核酸 (LNA)。在某些實施例中，gRNA 分子之 5' 端處的第一個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，該引導 RNA 分子的 5' 端處的第一個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子之 5' 端處的第一個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子之 5' 端處的第一個核苷酸為 LNA。在某些實施例中，gRNA 分子之 5' 端處的第一個核苷酸具有去氧核糖。In certain embodiments, the gRNA molecule disclosed herein includes one or more of a 2'-fluorine-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribonucleotide, and a locked nucleic acid (LNA) at its 5' end, for example, within the first 5 nucleotides at the 5' end. For example, but not by way of limitation, the first nucleotide at the 5' end of the gRNA molecule is a 2'-fluorine-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribonucleotide, or a locked nucleic acid (LNA). In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is a 2'-fluorine-modified nucleotide. In some embodiments, the first nucleotide at the 5' end of the guide RNA molecule is a 2'-O-methyl modified nucleotide. In some embodiments, the first nucleotide at the 5' end of the gRNA molecule is a 2'-O-(2-methoxyethyl) modified nucleotide. In some embodiments, the first nucleotide at the 5' end of the gRNA molecule is LNA. In some embodiments, the first nucleotide at the 5' end of the gRNA molecule has a deoxyribose sugar.

在某些實施例中，本揭露之 gRNA 在其 5' 端處，例如在 5' 端處的前 5 個核苷酸內包括兩個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA。例如但不作為限制，gRNA 分子之 5' 端處的第一個與第二個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之 5' 端處的第一個與第二個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子的末端處的第一個與第二個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子的 5' 端處的第一個與第二個核苷酸為 LNA。在某些實施例中，gRNA 分子的 5' 端處的第一個與第二個核苷酸具有去氧核糖。In certain embodiments, the gRNA disclosed herein comprises two or more 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 5' end, for example, within the first 5 nucleotides at the 5' end. For example, but not by way of limitation, the first and second nucleotides at the 5' end of the gRNA molecule are 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, LNAs or a combination thereof. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are 2'-fluorine-modified nucleotides. In some embodiments, the first and second nucleotides at the 5' end of the guide RNA molecule are 2'-O-methyl modified nucleotides. In some embodiments, the first and second nucleotides at the ends of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In some embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are LNA. In some embodiments, the first and second nucleotides at the 5' end of the gRNA molecule have deoxyribose.

在某些實施例中，本揭露之 gRNA 在其 5' 端處，例如在 5' 端處的前 5 個核苷酸內包括三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA。例如但不作為限制，gRNA 分子之 5' 端處的第一個、第二個與第三個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之 5' 端處的第一個、第二個與第三個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，該 gRNA 分子的 5' 端處的第一個、第二個與第三個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子的末端處的第一個、第二個與第三個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個與第三個核苷酸為 LNA。在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個與第三個核苷酸具有去氧核糖。In certain embodiments, the gRNA disclosed herein comprises three or more 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 5' end, for example, within the first 5 nucleotides at the 5' end. For example, but not by way of limitation, the first, second and third nucleotides at the 5' end of the gRNA molecule are 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, LNAs or a combination thereof. In certain embodiments, the first, second and third nucleotides at the 5' end of the gRNA molecule are 2'-fluorine-modified nucleotides. In some embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl modified nucleotides. In some embodiments, the first, second, and third nucleotides at the end of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In some embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are LNA. In some embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule have deoxyribose.

在某些實施例中，本揭露之 gRNA 在其 5' 端處，例如在 5' 端處的前 5 個核苷酸內包括四個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA。例如但不作為限制，gRNA 分子之 5' 端處的第一個、第二個、第三個與第四個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之 5' 端處的第一個、第二個、第三個與第四個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，該 gRNA 分子的 5' 端處的第一個、第二個、第三個與第四個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子的末端處的第一個、第二個、第三個與第四個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個、第三個與第四個核苷酸為 LNA。在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個、第三個與第四個核苷酸具有去氧核糖。In certain embodiments, the gRNA disclosed herein comprises four or more 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 5' end, for example, within the first 5 nucleotides at the 5' end. For example, but not by way of limitation, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, LNAs, or a combination thereof. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are 2'-fluorine-modified nucleotides. In some embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl modified nucleotides. In some embodiments, the first, second, third, and fourth nucleotides at the ends of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In some embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are LNA. In some embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule have deoxyribose.

在某些實施例中，本揭露之 gRNA 在其 5' 端處，例如在 5' 端處的前 5 個核苷酸內包括五個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA。例如但不作為限制，gRNA 分子之 5' 端處的第一個、第二個、第三個、第四個與第五個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之 5' 端處的第一個、第二個、第三個、第四個與第五個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，該 gRNA 分子的 5' 端處的第一個、第二個、第三個、第四個與第五個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子的末端處的第一個、第二個、第三個、第四個與第五個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個、第三個、第四個與第五個核苷酸為 LNA。在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個、第三個、第四個與第五個核苷酸具有去氧核糖。In certain embodiments, the gRNA disclosed herein comprises five or more 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 5' end, for example, within the first 5 nucleotides at the 5' end. For example, but not by way of limitation, the first, second, third, fourth and fifth nucleotides at the 5' end of the gRNA molecule are 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, LNAs or a combination thereof. In certain embodiments, the first, second, third, fourth and fifth nucleotides at the 5' end of the gRNA molecule are 2'-fluorine-modified nucleotides. In some embodiments, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl modified nucleotides. In some embodiments, the first, second, third, fourth, and fifth nucleotides at the end of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In some embodiments, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are LNA. In some embodiments, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule have deoxyribose.

在某些實施例中，本揭露之 gRNA 在其 3' 端處，例如在 3' 端處的最後 5 個核苷酸內包括經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或鎖核酸 (LNA) 中之一或多個。例如但不作為限制，gRNA 分子的 3' 端 (第「n」個) 核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或鎖核酸 (LNA)。在某些實施例中，gRNA 分子之第 n 個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，gRNA 分子之第 n 個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子之第 n 個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子之第 n 個核苷酸為 LNA。在某些實施例中，gRNA 分子之第 n 個核苷酸具有去氧核糖。In certain embodiments, the gRNA disclosed herein comprises one or more of a 2'-fluorine-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribonucleotide, and/or a locked nucleic acid (LNA) at its 3' end, such as within the last 5 nucleotides at the 3' end. For example, but not by way of limitation, the 3'-end ("nth") nucleotide of the gRNA molecule is a 2'-fluorine-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribonucleotide, or a locked nucleic acid (LNA). In certain embodiments, the nth nucleotide of the gRNA molecule is a 2'-fluorine-modified nucleotide. In some embodiments, the nth nucleotide of the gRNA molecule is a 2'-O-methyl modified nucleotide. In some embodiments, the nth nucleotide of the gRNA molecule is a 2'-O-(2-methoxyethyl) modified nucleotide. In some embodiments, the nth nucleotide of the gRNA molecule is LNA. In some embodiments, the nth nucleotide of the gRNA molecule has a deoxyribose sugar.

在某些實施例中，本揭露之 gRNA 在其 3' 端處，例如在 3' 端處的最後 5 個核苷酸內包括兩個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA。例如但不作為限制，gRNA 分子之第 n 個與第 n-1 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之第 n 個與第 n-1 個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，引導 RNA 分子之第 n 個與第 n-1 個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子的末端處的第 n 個與第 n-1 個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子之第 n 個與第 n-1 個核苷酸為 LNA。在某些實施例中，gRNA 分子之第 n 個與第 n-1 個核苷酸具有去氧核糖。In certain embodiments, the gRNA disclosed herein comprises two or more 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 3' end, for example, within the last 5 nucleotides at the 3' end. For example, but not by way of limitation, the nth and n-1th nucleotides of the gRNA molecule are 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, LNAs or a combination thereof. In certain embodiments, the nth and n-1th nucleotides of the gRNA molecule are 2'-fluorine-modified nucleotides. In some embodiments, the nth and n-1th nucleotides of the guide RNA molecule are 2'-O-methyl modified nucleotides. In some embodiments, the nth and n-1th nucleotides at the ends of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In some embodiments, the nth and n-1th nucleotides of the gRNA molecule are LNA. In some embodiments, the nth and n-1th nucleotides of the gRNA molecule have deoxyribose.

在某些實施例中，本揭露之 gRNA 在其 3' 端處，例如在 3' 端處的最後 5 個核苷酸內包括三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA。例如但不作為限制，gRNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，引導 RNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子的末端處的第 n 個、第 n-1 個與第 n-2 個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸為 LNA。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸具有去氧核糖。In certain embodiments, the gRNA disclosed herein comprises three or more 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 3' end, for example, within the last 5 nucleotides at the 3' end. For example, but not by way of limitation, the nth, n-1th, and n-2th nucleotides of the gRNA molecule are 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, LNAs, or a combination thereof. In certain embodiments, the nth, n-1th, and n-2th nucleotides of the gRNA molecule are 2'-fluorine-modified nucleotides. In some embodiments, the nth, n-1th, and n-2th nucleotides of the guide RNA molecule are 2'-O-methyl modified nucleotides. In some embodiments, the nth, n-1th, and n-2th nucleotides at the ends of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In some embodiments, the nth, n-1th, and n-2th nucleotides of the gRNA molecule are LNA. In some embodiments, the nth, n-1th, and n-2th nucleotides of the gRNA molecule have deoxyribose.

在某些實施例中，本揭露之 gRNA 在其 3' 端處，例如在 3' 端處的最後 5 個核苷酸內包括四個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA。例如但不作為限制，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，引導 RNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子的末端處的第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸為 LNA。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸具有去氧核糖。在某些實施例中，gRNA 分子之第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為經 2'-O-甲基修飾的核苷酸。In certain embodiments, the gRNA disclosed herein comprises four or more 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 3' end, for example, within the last 5 nucleotides at the 3' end. For example, but not by way of limitation, the nth, n-1th, n-2th and n-3th nucleotides of the gRNA molecule are 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, LNAs or a combination thereof. In certain embodiments, the n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, LNA, or a combination thereof. In certain embodiments, the n-1, n-2, and n-3 nucleotides of the gRNA molecule are 2'-fluorine-modified nucleotides. In certain embodiments, the n-1, n-2, and n-3 nucleotides of the guide RNA molecule are 2'-O-methyl-modified nucleotides. In some embodiments, the nth, n-1th, n-2th, and n-3th nucleotides at the ends of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In some embodiments, the nth, n-1th, n-2th, and n-3th nucleotides of the gRNA molecule are LNA. In some embodiments, the nth, n-1th, n-2th, and n-3th nucleotides of the gRNA molecule have deoxyribose. In some embodiments, the n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule are 2'-O-methyl modified nucleotides.

在某些實施例中，本揭露之 gRNA 在其 3' 端處，例如在 3' 端處的最後 5 個核苷酸內包括五個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA。例如但不作為限制，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸、LNA 或其組合。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為經 2'-氟修飾的核苷酸。在某些實施例中，引導 RNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為經 2'-O-甲基修飾的核苷酸。在某些實施例中，gRNA 分子的末端處的第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為 LNA。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸具有去氧核糖。In certain embodiments, the gRNA disclosed herein comprises five or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 3' end, for example, within the last 5 nucleotides at the 3' end. For example, but not by way of limitation, the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, LNAs or a combination thereof. In some embodiments, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule are 2'-fluorine-modified nucleotides. In some embodiments, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides of the guide RNA molecule are 2'-O-methyl-modified nucleotides. In some embodiments, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides at the ends of the gRNA molecule are 2'-O-(2-methoxyethyl)-modified nucleotides. In some embodiments, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule are LNAs. In certain embodiments, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule have deoxyribose.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 5' 端處的一或多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 5' 端處的一或多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之 5' 端處的第一個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或鎖核酸 (LNA)，且 gRNA 包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之 5' 端處的第一個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises (i) one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, and/or LNAs at its 5' end, and (ii) one or more phosphorodithioate bonds at the 5' end of the gRNA molecule. In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribonucleotide, or a locked nucleic acid (LNA), and the gRNA comprises a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is a 2'-O-methyl modified nucleotide, and the gRNA includes a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 5' 端處的兩個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 5' 端處的兩個或更多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之 5' 端處的第一個與第二個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或 LNA，且 gRNA 分子包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸以及第二個與第三個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之 5' 端處的第一個與第二個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 分子包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸以及第二個與第三個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure includes (i) two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, and/or LNAs at its 5' end, and (ii) two or more phosphorodithioate bonds at the 5' end of the gRNA molecule. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, or LNAs, and the gRNA molecule includes phosphorodithioate bonds between the first and second nucleotides and the second and third nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl modified nucleotides, and the gRNA molecule includes phosphorodithioate bonds between the first and second nucleotides and the second and third nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 5' 端處的三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 5' 端處的三個或更多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之 5' 端處的第一個、第二個與第三個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或 LNA，且 gRNA 包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之 5' 端處的第一個、第二個與第三個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, and/or LNAs at its 5' end, and (ii) three or more phosphorodithioate bonds at the 5' end of the gRNA molecule. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, or LNAs, and the gRNA comprises phosphorodithioate bonds between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl modified nucleotides, and the gRNA includes phosphorodithioate bonds between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 5' 端處的四個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 5' 端處的四個或更多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之 5' 端處的第一個、第二個、第三個與第四個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或 LNA，且 gRNA 包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、第三個與第四個核苷酸以及第四個與第五個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之 5' 端處的第一個、第二個、第三個與第四個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 包括在 gRNA 分子之 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、第三個與第四個核苷酸以及第四個與第五個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises (i) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 5' end, and (ii) four or more phosphorodithioate bonds at the 5' end of the gRNA molecule. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, or LNAs, and the gRNA includes phosphorodithioate bonds between the first and second nucleotides, the second and third nucleotides, the third and fourth nucleotides, and the fourth and fifth nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl-modified nucleotides, and the gRNA includes phosphorodithioate bonds between the first and second nucleotides, the second and third nucleotides, the third and fourth nucleotides, and the fourth and fifth nucleotides at the 5' end of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 3' 端處的一或多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 3' 端處的一或多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或鎖核酸 (LNA)，且 gRNA 包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises (i) one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, and/or LNAs at its 3' end, and (ii) one or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the nth nucleotide of the gRNA molecule is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribonucleotide, or a locked nucleic acid (LNA), and the gRNA comprises a phosphorodithioate bond between the nth and n-1th nucleotides of the gRNA molecule. In certain embodiments, the nth nucleotide of the gRNA molecule is a 2'-O-methyl modified nucleotide, and the gRNA comprises a phosphorodithioate bond between the nth and n-1th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 3' 端處的兩個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 3' 端處的兩個或更多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個與第 n-1 個、或第 n 個與第 n-2 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸或 LNA，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸之間或第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個與第 n-1 個核苷酸或第 n 個與第 n-2 個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸之間、或第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises (i) two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 3' end, and (ii) two or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the nth and n-1th, or the nth and n-2th nucleotides of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, or LNAs, and the gRNA molecule comprises a phosphorodithioate bond between the nth and n-1th nucleotides and the n-1th and n-2th nucleotides or between the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the gRNA molecule. In certain embodiments, the nth and n-1th nucleotides or the nth and n-2th nucleotides of the gRNA molecule are 2'-O-methyl modified nucleotides, and the gRNA molecule comprises a phosphorothioate bond between the nth and n-1th nucleotides and the n-1th and n-2th nucleotides, or between the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 3' 端處的三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 3' 端處的兩個或更多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基)修飾的核苷酸、去氧核糖核苷酸或 LNA，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸之間、或第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸之間、或第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個與第 n-2 個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸之間以及第 n-1 個與第 n-2 個核苷酸之間、或第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 3' end, and (ii) two or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the nth, n-1th, and n-2th nucleotides of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, or LNAs, and the gRNA molecule comprises a phosphorodithioate bond between the nth and n-1th nucleotides and the n-1th and n-2th nucleotides, or between the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the gRNA molecule. In certain embodiments, the nth, n-1th, and n-2th nucleotides of the gRNA molecule are 2'-O-methyl modified nucleotides, and the gRNA molecule includes a phosphorodithioate bond between the nth and n-1th nucleotides and the n-1th and n-2th nucleotides, or between the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the gRNA molecule. In certain embodiments, the nth, n-1th, and n-2th nucleotides of the gRNA molecule are 2'-O-methyl modified nucleotides, and the gRNA molecule includes a phosphorodithioate bond between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides, or between the nth and n-1th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 3' 端處的四個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 3' 端處的四個或更多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或 LNA，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間、第 n-2 個與第 n-3 個核苷酸之間以及第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間、第 n-2 個與第 n-3 個核苷酸之間以及第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises (i) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 3' end, and (ii) four or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the nth, n-1th, n-2th, and n-3th nucleotides of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, or LNAs, and the gRNA molecule comprises phosphorodithioate bonds between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, between the n-2th and n-3th nucleotides, and between the n-3th and n-4th nucleotides of the gRNA molecule. In certain embodiments, the nth, n-1th, n-2th, and n-3th nucleotides of the gRNA molecule are 2'-O-methyl modified nucleotides, and the gRNA molecule comprises phosphorodithioate bonds between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, between the n-2th and n-3th nucleotides, and between the n-3th and n-4th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括 (i) 在其 3' 端處的五個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，以及 (ii) 在 gRNA 分子之 3' 端處的四個或更多個二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸或 LNA，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間、第 n-2 個與第 n-3 個核苷酸之間以及第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸為經 2'-O-甲基修飾的核苷酸，且 gRNA 分子包括在 gRNA 分子之第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間、第 n-2 個與第 n-3 個核苷酸之間以及第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises (i) five or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at its 3' end, and (ii) four or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, or LNAs, and the gRNA molecule comprises phosphorodithioate bonds between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, between the n-2th and n-3th nucleotides, and between the n-3th and n-4th nucleotides of the gRNA molecule. In certain embodiments, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule are 2'-O-methyl modified nucleotides, and the gRNA molecule comprises phosphorodithioate bonds between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, between the n-2th and n-3th nucleotides, and between the n-3th and n-4th nucleotides of the gRNA molecule.

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的一或多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，(ii) 在 5' 端處的一或多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的一或多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及 LNA，以及 (iv) 在 3' 端處的一或多個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的一或多個經 2'-O-甲基修飾的核苷酸，(ii) 在 5' 端處的一或多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的一或多個經 2'-O-甲基修飾的核苷酸，以及 (iv) 在 3' 端處的一或多個二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises: (i) one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at the 5' end, (ii) one or more phosphorodithioate bonds at the 5' end, (iii) one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and LNAs at the 3' end, and (iv) one or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the present disclosure comprises: (i) one or more 2'-O-methyl modified nucleotides at the 5' end, (ii) one or more phosphorodithioate bonds at the 5' end, (iii) one or more 2'-O-methyl modified nucleotides at the 3' end, and (iv) one or more phosphorodithioate bonds at the 3' end.

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的兩個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，(ii) 在 5' 端處的兩個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的兩個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及 LNA，以及 (iv) 在 3' 端處的兩個或更多個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的兩個或更多個經 2'-O-甲基修飾的核苷酸，(ii) 在 5' 端處的兩個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的兩個或更多個經 2'-O-甲基修飾的核苷酸，以及 (iv) 在 3' 端處的兩個或更多個二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises: (i) two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at the 5' end, (ii) two or more phosphorodithioate bonds at the 5' end, (iii) two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and LNAs at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the present disclosure comprises: (i) two or more 2'-O-methyl modified nucleotides at the 5' end, (ii) two or more phosphorodithioate bonds at the 5' end, (iii) two or more 2'-O-methyl modified nucleotides at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end.

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，(ii) 在 5' 端處的兩個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及 LNA，以及 (iv) 在 3' 端處的兩個或更多個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個或更多個經 2'-O-甲基修飾的核苷酸，(ii) 在 5' 端處的兩個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的三個或更多個經 2'-O-甲基修飾的核苷酸，以及 (iv) 在 3' 端處的兩個或更多個二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises: (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at the 5' end, (ii) two or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and LNAs at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the present disclosure comprises: (i) three or more 2'-O-methyl modified nucleotides at the 5' end, (ii) two or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-O-methyl modified nucleotides at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end.

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，(ii) 在 5' 端處的三個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及 LNA，以及 (iv) 在 3' 端處的一或多個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個或更多個經 2'-O-甲基修飾的核苷酸，(ii) 在 5' 端處的三個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的三個或更多個經 2'-O-甲基修飾的核苷酸，以及 (iv) 在 3' 端處的一或多個二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises: (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at the 5' end, (ii) three or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and LNAs at the 3' end, and (iv) one or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the present disclosure comprises: (i) three or more 2'-O-methyl modified nucleotides at the 5' end, (ii) three or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-O-methyl modified nucleotides at the 3' end, and (iv) one or more phosphorodithioate bonds at the 3' end.

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，(ii) 在 5' 端處的三個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的三個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及 LNA，以及 (iv) 在 3' 端處的兩個或更多個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個或更多個經 2'-O-甲基修飾的核苷酸，(ii) 在 5' 端處的三個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的三個或更多個經 2'-O-甲基修飾的核苷酸，以及 (iv) 在 3' 端處的兩個或更多個二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises: (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at the 5' end, (ii) three or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and LNAs at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the present disclosure comprises: (i) three or more 2'-O-methyl modified nucleotides at the 5' end, (ii) three or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-O-methyl modified nucleotides at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end.

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的四個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及/或 LNA，(ii) 在 5' 端處的四個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的四個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及 LNA，以及 (iv) 在 3' 端處的四個或更多個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的四個或更多個經 2'-O-甲基修飾的核苷酸，(ii) 在 5' 端處的四個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的四個或更多個經 2'-O-甲基修飾的核苷酸，以及 (iv) 在 3' 端處的四個或更多個二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises: (i) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and/or LNAs at the 5' end, (ii) four or more phosphorodithioate bonds at the 5' end, (iii) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides and LNAs at the 3' end, and (iv) four or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the present disclosure comprises: (i) four or more 2'-O-methyl modified nucleotides at the 5' end, (ii) four or more phosphorodithioate bonds at the 5' end, (iii) four or more 2'-O-methyl modified nucleotides at the 3' end, and (iv) four or more phosphorodithioate bonds at the 3' end.

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的四個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及 LNA，(ii) 在 5' 端處的四個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的五個或更多個經 2'-氟修飾的核苷酸、經 2'-O-甲基修飾的核苷酸、經 2'-O-(2-甲氧基乙基) 修飾的核苷酸、去氧核糖核苷酸及 LNA，以及 (iv) 在 3' 端處的四個或更多個二硫代磷酸酯鍵結。在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的四個或更多個經 2'-O-甲基修飾的核苷酸，(ii) 在 5' 端處的四個或更多個二硫代磷酸酯鍵結，(iii) 在 3' 端處的五個或更多個經 2'-O-甲基修飾的核苷酸，以及 (iv) 在 3' 端處的四個或更多個二硫代磷酸酯鍵結。In certain embodiments, the gRNA of the present disclosure comprises: (i) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, and LNAs at the 5' end, (ii) four or more phosphorodithioate bonds at the 5' end, (iii) five or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribonucleotides, and LNAs at the 3' end, and (iv) four or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the present disclosure comprises: (i) four or more 2'-O-methyl modified nucleotides at the 5' end, (ii) four or more phosphorodithioate bonds at the 5' end, (iii) five or more 2'-O-methyl modified nucleotides at the 3' end, and (iv) four or more phosphorodithioate bonds at the 3' end.

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的經 2'-O-甲基修飾的核苷酸 (例如，第一核苷酸)，(ii) 在 5' 端處的一個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間)，(iii) 在 3' 端處的一個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個核苷酸)，以及 (iv) 在 3' 端處的一個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間)。In certain embodiments, the gRNA disclosed herein comprises: (i) a 2'-O-methyl modified nucleotide at the 5' end (e.g., the first nucleotide), (ii) a phosphorodithioate bond at the 5' end (e.g., between the first and second nucleotides), (iii) a 2'-O-methyl modified nucleotide at the 3' end (e.g., the nth nucleotide), and (iv) a phosphorodithioate bond at the 3' end (e.g., between the nth and n-1th nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的兩個經 2'-O-甲基修飾的核苷酸 (例如，第一個與第二個核苷酸)，(ii) 在 5' 端處的兩個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間以及第二個與第三個核苷酸之間)，(iii) 在 3' 端處的兩個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個與第 n-1 個核苷酸)，以及 (iv) 在 3' 端處的兩個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間以及第 n-1 個與第 n-2 個核苷酸之間)。In certain embodiments, the gRNA disclosed herein comprises: (i) two 2'-O-methyl modified nucleotides at the 5' end (e.g., the first and second nucleotides), (ii) two phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides and between the second and third nucleotides), (iii) two 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth and n-1th nucleotides), and (iv) two phosphorodithioate bonds at the 3' end (e.g., between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個與第三個核苷酸)，(ii) 在 5' 端處的三個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間、第二個與第三個核苷酸之間以及第三個與第四個核苷酸之間)，(iii) 在 3' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個與第 n-2 個核苷酸)，以及 (iv) 在 3' 端處的兩個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間以及第 n-1 個與第 n-2 個核苷酸之間)。In certain embodiments, the gRNA disclosed herein comprises: (i) three 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) three phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, and n-2th nucleotides), and (iv) two phosphorodithioate bonds at the 3' end (e.g., between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個與第三個核苷酸)，(ii) 在 5' 端處的三個硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間、第二個與第三個核苷酸之間以及第三個與第四個核苷酸之間)，(iii) 在 3' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個與第 n-2 個核苷酸)，以及 (iv) 在 3' 端處的兩個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間以及第 n-1 個與第 n-2 個核苷酸之間)。In certain embodiments, the gRNA disclosed herein comprises: (i) three 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) three phosphorothioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, and n-2th nucleotides), and (iv) two phosphorodithioate bonds at the 3' end (e.g., between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個與第三個核苷酸)，(ii) 在 5' 端處的兩個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間以及第二個與第三個核苷酸之間、或第二個與第三個核苷酸以及第三個與第四個核苷酸之間)，(iii) 在 3' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個與第 n-2 個核苷酸)，以及 (iv) 在 3' 端處的兩個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間以及第 n-1 個與第 n-2 個核苷酸之間、或第 n-1 個與第 n-2 個核苷酸之間以及第 n-2 個與第 n-3 個核苷酸之間)。In certain embodiments, the gRNA disclosed herein comprises: (i) three 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) two phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides and between the second and third nucleotides, or between the second and third nucleotides and between the third and fourth nucleotides), (iii) three 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, and n-2th nucleotides), and (iv) two phosphorodithioate bonds at the 3' end (e.g., between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides, or between the n-1th and n-2th nucleotides and between the n-1th and n-2th nucleotides). between the n-2nd and n-3rd nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個與第三個核苷酸)，(ii) 在 5' 端處的兩個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間以及第二個與第三個核苷酸之間、或第二個與第三個核苷酸以及第三個與第四個核苷酸之間)，(iii) 在 5' 端處的一個硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間、第二個與第三個核苷酸之間或第三個與第四個核苷酸之間)，(iv) 在 3' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個與第 n-2 個核苷酸)，以及 (v) 在 3' 端處的兩個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間以及第 n-1 個與第 n-2 個核苷酸之間、或第 n-1 個與第 n-2 個核苷酸之間以及第 n-2 個與第 n-3 個核苷酸之間)。In certain embodiments, the gRNA disclosed herein comprises: (i) three 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) two phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides and between the second and third nucleotides, or between the second and third nucleotides and between the third and fourth nucleotides), (iii) one phosphorothioate bond at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, or between the third and fourth nucleotides), (iv) three 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, and n-2th nucleotides), and (v) two phosphorodithioate bonds at the 3' end (e.g., between the nth and n-1th nucleotides and between the n-1th and n-2th nucleotides, or between the n-1th and n-2th nucleotides and between the n-2th and n-3th nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個與第三個核苷酸)，(ii) 在 5' 端處的三個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間、第二個與第三個核苷酸之間以及第三個與第四個核苷酸之間)，(iii) 在 3' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個與第 n-2 個核苷酸)，以及 (iv) 在 3' 端處的一個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間或第 n-2 個與第 n-3 個核苷酸之間)。In certain embodiments, the gRNA disclosed herein comprises: (i) three 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) three phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, and n-2th nucleotides), and (iv) one phosphorodithioate bond at the 3' end (e.g., between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, or between the n-2th and n-3th nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個與第三個核苷酸)，(ii) 在 5' 端處的三個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間、第二個與第三個核苷酸之間以及第三個與第四個核苷酸之間)，(iii) 在 3' 端處的三個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個與第 n-2 個核苷酸)，(iv) 在 3' 端處的一個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間或第 n-2 個與第 n-3 個核苷酸之間)，以及 (v) 在 3' 端處的一個硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間或第 n-2 個與第 n-3 個核苷酸之間)。In certain embodiments, the gRNA of the present disclosure comprises: (i) three 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) three phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, and n-2th nucleotides), (iv) one phosphorodithioate bond at the 3' end (e.g., between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, or between the n-2th and n-3th nucleotides), and (v) one phosphorodithioate bond at the 3' end. a phosphorothioate bond at the end of the nucleotide (e.g., between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, or between the n-2th and n-3th nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的四個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個、第三個與第四個核苷酸)，(ii) 在 5' 端處的四個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間、第二個與第三個核苷酸之間、第三個與第四個核苷酸之間以及第四個與第五個核苷酸之間)，(iii) 在 3' 端處的四個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸)，以及 (iv) 在 3' 端處的四個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間、第 n-2 個與第 n-3 個核苷酸之間以及第 n-3 個與第 n-4 個核苷酸之間)。In certain embodiments, the gRNA of the present disclosure comprises: (i) four 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, third, and fourth nucleotides), (ii) four phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, and between the fourth and fifth nucleotides), (iii) four 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, n-2th, and n-3th nucleotides), and (iv) four phosphorodithioate bonds at the 3' end (e.g., between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, between the n-2th and n-3th nucleotides). between nucleotides n-3 and between nucleotides n-3 and n-4).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的四個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個、第三個與第四個核苷酸)，(ii) 在 5' 端處的四個二硫代磷酸酯鍵結 (例如，第一個與第二個核苷酸之間、第二個與第三個核苷酸之間、第三個與第四個核苷酸之間以及第四個與第五個核苷酸之間)，(iii) 在 3' 端處的五個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸)，以及 (iv) 在 3' 端處的四個二硫代磷酸酯鍵結 (例如，第 n 個與第 n-1 個核苷酸之間、第 n-1 個與第 n-2 個核苷酸之間、第 n-2 個與第 n-3 個核苷酸之間以及第 n-3 個與第 n-4 個核苷酸之間)。In certain embodiments, the gRNA of the present disclosure comprises: (i) four 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, third, and fourth nucleotides), (ii) four phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, and between the fourth and fifth nucleotides), (iii) five 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, n-2th, n-3th, and n-4th nucleotides), and (iv) four phosphorodithioate bonds at the 3' end (e.g., between the nth and n-1th nucleotides, between the n-1th and n-2th nucleotides, between the n-3th and n-4th nucleotides). between the n-2nd and n-3rd nucleotides and between the n-3rd and n-4th nucleotides).

在某些實施例中，本揭露之 gRNA 包括：(i) 在 5' 端處的四個經 2'-O-甲基修飾的核苷酸 (例如，第一個、第二個、第三個與第四個核苷酸)，(ii) 在 5' 端處的四個二硫代磷酸酯鍵結 (例如，第一個與第二核苷酸之間、第二個與第三個核苷酸之間、第三個與第四個核苷酸之間以及第四個與第五個核苷酸之間)，(iii) 在 3' 端處的五個經 2'-O-甲基修飾的核苷酸 (例如，第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸)，以及 (iv) 在 3' 端處的四個二硫代磷酸酯鍵結 (例如，第 n-1 個與第 n-2 個核苷酸之間、第 n-2 個與第 n-3 個核苷酸之間、第 n-3 個與第 n-4 個核苷酸之間以及第 n-4 個與第 n-5 個核苷酸之間)。In certain embodiments, the gRNA of the present disclosure comprises: (i) four 2'-O-methyl modified nucleotides at the 5' end (e.g., the first, second, third, and fourth nucleotides), (ii) four phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, and between the fourth and fifth nucleotides), (iii) five 2'-O-methyl modified nucleotides at the 3' end (e.g., the nth, n-1th, n-2th, n-3th, and n-4th nucleotides), and (iv) four phosphorodithioate bonds at the 3' end (e.g., between the n-1th and n-2th nucleotides, between the n-2th and n-3th nucleotides, between the n-4th and n-5th nucleotides). between the n-3rd and n-4th nucleotides and between the n-4th and n-5th nucleotides).

在某些實施例中，本揭露之經修飾之 gRNA 可以根據表 1 及表 10 中所揭示之修飾方案進行修飾。例如但不作為限制，經修飾之 gRNA 可以根據表 1 之修飾方案「A」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「A」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「B」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「C」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「D」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「E」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「F」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「G」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「H」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「I」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「J」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「K」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 1 之修飾方案「L」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「M」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「N」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「O」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「P」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「Q」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「R」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「S」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「T」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「U」進行修飾。在某些實施例中，經修飾之 gRNA 可以根據表 10 之修飾方案「V」進行修飾。In certain embodiments, the modified gRNA of the present disclosure may be modified according to the modification schemes disclosed in Table 1 and Table 10. For example, but not by way of limitation, the modified gRNA may be modified according to the modification scheme "A" of Table 1. In certain embodiments, the modified gRNA may be modified according to the modification scheme "A" of Table 1. In certain embodiments, the modified gRNA may be modified according to the modification scheme "B" of Table 1. In certain embodiments, the modified gRNA may be modified according to the modification scheme "C" of Table 1. In certain embodiments, the modified gRNA may be modified according to the modification scheme "D" of Table 1. In certain embodiments, the modified gRNA may be modified according to the modification scheme "E" of Table 1. In certain embodiments, the modified gRNA may be modified according to modification scheme "F" of Table 1. In certain embodiments, the modified gRNA may be modified according to modification scheme "G" of Table 1. In certain embodiments, the modified gRNA may be modified according to modification scheme "H" of Table 1. In certain embodiments, the modified gRNA may be modified according to modification scheme "I" of Table 1. In certain embodiments, the modified gRNA may be modified according to modification scheme "J" of Table 1. In certain embodiments, the modified gRNA may be modified according to modification scheme "K" of Table 1. In certain embodiments, the modified gRNA may be modified according to modification scheme "L" of Table 1. In certain embodiments, the modified gRNA may be modified according to the modification scheme "M" of Table 10. In certain embodiments, the modified gRNA may be modified according to the modification scheme "N" of Table 10. In certain embodiments, the modified gRNA may be modified according to the modification scheme "O" of Table 10. In certain embodiments, the modified gRNA may be modified according to the modification scheme "P" of Table 10. In certain embodiments, the modified gRNA may be modified according to the modification scheme "Q" of Table 10. In certain embodiments, the modified gRNA may be modified according to the modification scheme "R" of Table 10. In certain embodiments, the modified gRNA may be modified according to the modification scheme "S" of Table 10. In certain embodiments, the modified gRNA may be modified according to modification scheme "T" of Table 10. In certain embodiments, the modified gRNA may be modified according to modification scheme "U" of Table 10. In certain embodiments, the modified gRNA may be modified according to modification scheme "V" of Table 10.

在某些實施例中，本揭露之經修飾之 gRNA 可具有以下的編輯效率：約 1%、約 2%、約 3%、約 4%、約 5%、約 6%、約 7%、約 8%、約 9%、約 10%、約 11%、約 12%、約 13%、約 14%、約 15%、約 16%、約 17%、約 18%、約 19%、約 20%、約 21%、約 22%、約 23%、約 24%、約 25%、約 26%、約 27%、約 28%、約 29%、約 30%、約 31%、約 32%、約 33%、約 34%、約 35%、約 36%、約 37%、約 38%、約 39%、約 40%、約 41%、約 42%、約 43%、約 44%、約 45%、約 46%、約 47%、約 48%、約 49%、約 50%、約 51%、約 52%、約 53%、約 54%、約 55%、約 56%、約 57%、約 58%、約 59%、約 60%、約 61%、約 62%、約 63%、約 64%、約 65%、約 66%、約 67%、約 68%、約 69%、約 70%、約 71%、約 72%、約 73%、約 74%、約 75%、約 76%、約 77%、約 78%、約 79%、約 80%、約 81%、約 82%、約 83%、約 84%、約 85%、約 86%、約 87%、約 88%、約 89%、約 90%、約 91%、約 92%、約 93%、約 94%、約 95%、約 96%、約 97%、約 98%、約 99% 或約 100%。In certain embodiments, the modified gRNA disclosed herein may have an editing efficiency of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

在某些實施例中，本揭露之經修飾之 gRNA 可具有以下的編輯效率：約 5% 或更高、約 10% 或更高、約 15% 或更高、約 20% 或更高、約 25% 或更高、約 30% 或更高、約 35% 或更高、約 40% 或更高、約 45% 或更高、約 50% 或更高、約 55% 或更高、約 60% 或更高、約 65% 或更高、約 70% 或更高、約 75% 或更高、約 80% 或更高、約 85% 或更高、約 90% 或更高或約 95% 或更高。在某些實施例中，本揭露之經修飾之 gRNA 具有約 60% 或更高的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 具有約 65% 或更高的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 具有約 70% 或更高的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 具有約 75% 或更高的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 具有約 80% 或更高的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 具有約 85% 或更高的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 具有約 90% 或更高的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 具有約 95% 或更高的編輯效率。In certain embodiments, the modified gRNA of the present disclosure may have an editing efficiency of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more. In certain embodiments, the modified gRNA of the present disclosure has an editing efficiency of about 60% or more. In certain embodiments, the modified gRNA of the present disclosure has an editing efficiency of about 65% or more. In certain embodiments, the modified gRNA of the present disclosure has an editing efficiency of about 70% or more. In certain embodiments, the modified gRNA of the present disclosure has an editing efficiency of about 75% or more. In certain embodiments, the modified gRNA of the present disclosure has an editing efficiency of about 80% or more. In certain embodiments, the modified gRNA of the present disclosure has an editing efficiency of about 85% or more. In certain embodiments, the modified gRNA of the present disclosure has an editing efficiency of about 90% or more. In certain embodiments, the modified gRNA of the present disclosure has an editing efficiency of about 95% or more.

在某些實施例中，本揭露之經修飾之 gRNA 可具有約 5% 至約 100% 的編輯效率。例如但不作為限制，本揭露之經修飾之 gRNA 可具有以下的編輯效率：約 5% 至約 100%、約 10% 至約 100%、約 20% 至約 100%、約 30% 至約 100%、約 40% 至約 100%、約 45% 至約 100%、約 50% 至約 100%、約 55% 至約 100%、約 60% 至約 100%、約 65% 至約 100%、約 70% 至約 100%、約 75% 至約 100%、約 80% 至約 100%、約 85% 至約 100%、約 90% 至約 100% 或約 95% 至約 100%。在某些實施例中，本揭露之經修飾之 gRNA 可具有約 50% 至約 100% 的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 可具有約 60% 至約 100% 的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 可具有約 70% 至約 100% 的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 可具有約 80% 至約 100% 的編輯效率。在某些實施例中，本揭露之經修飾之 gRNA 可具有約 90% 至約 100% 的編輯效率。In certain embodiments, the modified gRNA disclosed herein may have an editing efficiency of about 5% to about 100%. For example, but not by way of limitation, the modified gRNA of the present disclosure may have an editing efficiency of about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100%. In certain embodiments, the modified gRNA of the present disclosure may have an editing efficiency of about 50% to about 100%. In certain embodiments, the modified gRNA of the present disclosure may have an editing efficiency of about 60% to about 100%. In certain embodiments, the modified gRNA of the present disclosure may have an editing efficiency of about 70% to about 100%. In certain embodiments, the modified gRNA of the present disclosure may have an editing efficiency of about 80% to about 100%. In certain embodiments, the modified gRNA of the present disclosure may have an editing efficiency of about 90% to about 100%.

在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率或具有與參考 gRNA 相比改善的編輯效率。例如但不作為限制，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 50% 或更高、約 55% 或更高、約 60% 或更高、約 65% 或更高、約 70% 或更高、約 75% 或更高、約 80% 或更高、約 85% 或更高、約 90% 或更高或約 95% 或更高。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 50% 或更高。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 60% 或更高。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 70% 或更高。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 80% 或更高。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 90% 或更高。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 50% 至約 100%，例如約 55% 至約 100%、約 60% 至約 100%、約 65% 至約 100%、約 70% 至約 100%、約 75% 至約 100%、約 80% 至約 100%、約 85% 至約 100%、約 90% 至約 100%、約 91% 至約 100%、約 92% 至約 100%、約 93% 至約 100%、約 94% 至約 100%、約 95% 至約 100%、約 96% 至約 100%、約 97% 至約 100%、約 98% 至約 100% 或約 99% 至約 100%。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 60% 至約 100%。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 70% 至約 100%。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 80% 至約 100%。在某些實施例中，本揭露之經修飾之 gRNA 可以保留參考 gRNA 之編輯效率的約 90% 至約 100%。 在某些實施例中，與參考 gRNA 相比，本揭露之經修飾之 gRNA 可表現出編輯效率之增加，例如，編輯效率增加約 1% 或更高、約 2% 或更高、約 3% 或更高、約 4% 或更高、約 5% 或更高、約 6% 或更高、約 7% 或更高、約 8% 或更高、約 9% 或更高或約 10% 或更高。在某些實施例中，本揭露之經修飾之 gRNA 可表現出編輯效率自約 1% 至約 10% 之增加。在某些實施例中，參考 gRNA 可為具有與經修飾之 gRNA 相同的核苷酸序列之 gRNA (例如，沒有與經修飾之 gRNA 相同的修飾)。在某些實施例中，參考 gRNA 可為具有靶向域的 gRNA，該靶向域具有與經修飾之 gRNA 相同的序列 (例如，沒有與經修飾之 gRNA 相同的修飾)。在某些實施例中，參考 gRNA 可為具有與經修飾之 gRNA 相同數量之核苷酸的 gRNA (例如，沒有與經修飾之 gRNA 相同的修飾)。III. RNA引導的核酸酶In certain embodiments, the modified gRNA of the present disclosure may retain the editing efficiency of a reference gRNA or have an improved editing efficiency compared to a reference gRNA. For example, but not by way of limitation, the modified gRNA of the present disclosure may retain about 50% or more of the editing efficiency of a reference gRNA, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more. In certain embodiments, the modified gRNA of the present disclosure may retain about 50% or more of the editing efficiency of a reference gRNA. In certain embodiments, the modified gRNA of the present disclosure may retain about 60% or more of the editing efficiency of a reference gRNA. In certain embodiments, the modified gRNA of the present disclosure can retain about 70% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the present disclosure can retain about 80% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the present disclosure can retain about 90% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the present disclosure can retain about 50% to about 100% of the editing efficiency of the reference gRNA, such as about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%. In certain embodiments, the modified gRNA of the present disclosure can retain about 60% to about 100% of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the present disclosure can retain about 70% to about 100% of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the present disclosure can retain about 80% to about 100% of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the present disclosure can retain about 90% to about 100% of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the present disclosure may exhibit an increase in editing efficiency compared to a reference gRNA, for example, an increase in editing efficiency of about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, or about 10% or more. In certain embodiments, the modified gRNA of the present disclosure may exhibit an increase in editing efficiency from about 1% to about 10%. In certain embodiments, the reference gRNA may be a gRNA having the same nucleotide sequence as the modified gRNA (e.g., without the same modification as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA having a targeting domain that has the same sequence as the modified gRNA (e.g., without the same modifications as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA having the same number of nucleotides as the modified gRNA (e.g., without the same modifications as the modified gRNA).III. RNA-guided nucleases

多種類型及物種之 RNA 引導的核酸酶可用於本揭露內容。RNA 引導的核酸酶與本文 (例如，第 II 節) 所揭示之一或多個經修飾之 gRNA 組合使用。Various types and species of RNA-guided nucleases can be used in the present disclosure. RNA-guided nucleases are used in combination with one or more modified gRNAs disclosed herein (e.g., Section II).

在某些實施例中，RNA 引導的核酸酶為 Cas 蛋白。Cas 蛋白之非限制性實例揭示於 Makarova 及 Koonin, Methods Mol.Biol.1311:47-75 (2015) 中，該文獻之內容藉由引用全文併入本文。在某些實施例中，Cas 蛋白包括 Cas1、Cas2、Cas3、Cas3-HD、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Cas12a (Cpf1)、Cas13、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、CsaX、Csm2、Csm3、Csm4、Csm5、Csm6、Csn2、Csb1、Csb2、Csb3、Csx1、Csx3、Csx10、Csx14、Csx15、Csx16、Csx17、Csf1、Csf2、Csf3、Csf4、C2c1、C2c2 及 C2c3。在某些實施例中，該 Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12a、Cas13 及其組合。在某些實施例中，Cas 蛋白為 Cas9 蛋白。In certain embodiments, the RNA-guided nuclease is a Cas protein. Non-limiting examples of Cas proteins are disclosed in Makarova and Koonin, Methods Mol. Biol. 1311:47-75 (2015), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the Cas protein includes Cas1, Cas2, Cas3, Cas3-HD, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a (Cpf1), Cas13, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, CsaX, Csm2, Csm3, Csm4, Csm5, Csm6, Csn2, Csb1, Csb2, Csb3, Csx1, Csx3, Csx10, Csx14, Csx15, Csx16, Csx17, Csf1, Csf2, Csf3, Csf4, C2c1, C2c2, and C2c3. In some embodiments, the Cas protein is selected from the group consisting of: Cas9, Cas12a, Cas13, and combinations thereof. In some embodiments, the Cas protein is a Cas9 protein.

在某些實施例中，Cas 蛋白為與參考 Cas 蛋白 (例如野生型 Cas 蛋白) 不同的工程化 Cas 蛋白。在某些實施例中，參考 Cas 蛋白為天然存在的 Cas 蛋白。在某些實施例中，與參考 Cas 蛋白 (例如野生型 Cas 蛋白) 相比，Cas 蛋白包括一或多個胺基酸變異。在某些實施例中，工程化 Cas 蛋白保留或基本上保留參考 Cas 蛋白之核酸酶 (例如，核酸內切酶) 活性。在某些實施例中，工程化 Cas 蛋白保留參考 Cas 蛋白之至少約 70%、約 80%、約 90%、約 95% 或約 99% 核酸酶活性。在某些實施例中，工程化 Cas 蛋白不具有裂解活性或不具有顯著的裂解活性。在某些實施例中，Cas 蛋白可以缺乏裂解活性或具有顯著較低的裂解活性，例如，小於參考 Cas 蛋白之裂解活性的 20%、約 10%、約 5% 或約 1%。In some embodiments, the Cas protein is an engineered Cas protein that is different from a reference Cas protein (e.g., a wild-type Cas protein). In some embodiments, the reference Cas protein is a naturally occurring Cas protein. In some embodiments, the Cas protein comprises one or more amino acid variations compared to a reference Cas protein (e.g., a wild-type Cas protein). In some embodiments, the engineered Cas protein retains or substantially retains the nuclease (e.g., endonuclease) activity of the reference Cas protein. In some embodiments, the engineered Cas protein retains at least about 70%, about 80%, about 90%, about 95%, or about 99% of the nuclease activity of the reference Cas protein. In some embodiments, the engineered Cas protein has no cleavage activity or has no significant cleavage activity. In certain embodiments, a Cas protein can lack cleavage activity or have significantly lower cleavage activity, e.g., less than 20%, about 10%, about 5%, or about 1% of the cleavage activity of a reference Cas protein.

在某些實施例中，工程化 Cas 蛋白包括一或多個缺失，該一或多個缺失減小 Cas 蛋白之尺寸，同時至少部分保留 Cas 蛋白之核酸酶活性。在某些實施例中，工程化 Cas 蛋白之減小的尺寸可以允許用於遞送此類工程化 Cas 蛋白之方法具有靈活性。In certain embodiments, the engineered Cas protein comprises one or more deletions that reduce the size of the Cas protein while at least partially retaining the nuclease activity of the Cas protein. In certain embodiments, the reduced size of the engineered Cas protein can allow for flexibility in the methods used to deliver such engineered Cas proteins.

在某些實施例中，Cas 蛋白與本揭露之 gRNA 分子相互作用，且與 gRNA 分子一致地定位至包括標靶序列 (例如，與 gRNA 分子之序列互補的序列) 及 PAM 序列的標靶核酸。在某些實施例中，Cas 蛋白與標靶核酸相互作用且裂解標靶核酸之能力依賴於 PAM 序列。在某些實施例中，標靶核酸之裂解發生在 PAM 序列的上游。在某些實施例中，標靶核酸之裂解發生在 PAM 序列的下游。來自不同物種 (例如，細菌物種) 之 Cas 分子可以識別不同的 PAM 序列。In certain embodiments, the Cas protein interacts with the gRNA molecule of the present disclosure and is localized in concert with the gRNA molecule to a target nucleic acid comprising a target sequence(e.g., a sequence complementary to the sequence of the gRNA molecule) and a PAM sequence. In certain embodiments, the ability of the Cas protein to interact with the target nucleic acid and to cleave the target nucleic acid is dependent on the PAM sequence. In certain embodiments, cleavage of the target nucleic acid occurs upstream of the PAM sequence. In certain embodiments, cleavage of the target nucleic acid occurs downstream of the PAM sequence. Casmolecules from different species (e.g., bacterial species) can recognize different PAM sequences.

在某些實施例中，用於本揭露之 Cas 蛋白可以源自以下物種中之任一者：化膿鏈球菌 (Streptococcus pyogenes)、肺炎鏈球菌 (Streptococcus pneumoniae)、嗜熱鏈球菌 (Streptococcus thermophilus)、無乳鏈球菌 (Streptococcus agalactiae)、副血鏈球菌 (Streptococcus parasanguinis)、口腔鏈球菌 (Streptococcus oralis)、涎鏈球菌 (Streptococcus salivarius)、獼猴鏈球菌 (Streptococcus macacae)、壞乳鏈球菌 (Streptococcus dysgalactiae)、咽峽炎鏈球菌 (Streptococcus anginosus)、星座鏈球菌 (Streptococcus constellatus)、假豚鏈球菌 (Streptococcus pseudoporcinus)、變種鏈球菌 (Streptococcus mutans)、無害李氏菌 (Listeria innocua)、蜜蜂螺原體 (Spiroplasma apis)、蠅類螺原體 (Spiroplasma syrphidicola)、卡托氏卟啉單胞菌 (Porphyromonas catoniae)、中間普雷沃菌 (Prevotella intermedia)、索氏密螺旋體 (Treponema socranskii)、大芬戈爾德菌 (Finegoldia magna)、貝氏巴氏桿菌 (Pasteurella bettyae)、錫蒂亞橄欖形菌 (Olivibacter sitiensis)、膠黏石面單孢菌 (Epilithonimonas tenax)、運動海研站菌 (Mesonia mobilis)、胚芽乳酸桿菌 (Lactobacillus plantarum)、紅蝽菌科細菌 (Coriobacteriaceae bacterium)、茂盛歐陸森氏菌 (Olsenella profusa)、痰嗜血桿菌 (Haemophilus sputorum)、臘樣芽孢桿菌 (Bacillus cereus)、米勒海水桿菌 (Aquimarina muellen)、沼澤金黃桿菌 (Chryseobacterium palustre)、草溶性擬桿菌 (Bacteroides graminisolvens)、腦膜炎雙球菌 (Neisseria meningitidis)、新兇手弗朗西斯菌 (Francisella novicida)、皮特曼嗜血桿菌 (Haemophilus pittmaniae)、貝特巴氏桿菌 (Pasteurella bettyae)、冷水黃桿菌 (Flavobacterium frigidarium)、土壤黃桿菌 (Flavobacterium soli) 及/或齒垢螺旋體 (Treponema denticola)。In certain embodiments, the Cas protein used in the present disclosure can be derived from any of the following species:Streptococcus pyogenes,Streptococcus pneumoniae ,Streptococcus thermophilus ,Streptococcus agalactiae ,Streptococcus parasanguinis ,Streptococcus oralis, Streptococcus salivarius, Streptococcusmacacae ,Streptococcus dysgalactiae ,Streptococcus anginosus ,Streptococcus constellatus. ),Streptococcus pseudoporcinus ,Streptococcus mutans ,Listeria innocua ,Spiroplasma apis ,Spiroplasma syrphidicola ,Porphyromonas catoniae ,Prevotella intermedia ,Treponema socranskii , Finegoldiamagna ,Pasteurella bettyae ,Olivibacter sitiensis ,Epilithonimonas tenax ,Mesonia mobilis ,Lactobacillus plantarum ),Coriobacteriaceae bacterium ,Olsenella profusa ,Haemophilus sputorum ,Bacillus cereus ,Aquimarina muellen ,Chryseobacterium palustre ,Bacteroides graminisolvens ,Neisseria meningitidis ,Francisella novicida ,Haemophilus pittmaniae ,Pasteurella bettyae ,Flavobacterium frigidarium ),Flavobacterium soli and/orTreponema denticola .

在某些實施例中，用於本揭露之 Cas 蛋白引導在標靶核酸之位置處的一或兩條股之裂解。例如但不作為限制，用於本揭露之 Cas 蛋白引導在標靶核酸內之一或兩條股的裂解。替代地，Cas 蛋白引導在來自標靶核酸之約 500 個鹼基對內 (例如，約 400 個、約 300 個、約 200 個、約 100 個、約 80 個、約 60 個、約 40 個、約 20 個、約 10 個或約 5 個鹼基對內) 的一或兩條股的裂解。In certain embodiments, a Cas protein used in the present disclosure directs the cleavage of one or two strands at a location in a target nucleic acid. For example, but not by way of limitation, a Cas protein used in the present disclosure directs the cleavage of one or two strands within a target nucleic acid. Alternatively, a Cas protein directs the cleavage of one or two strands within about 500 base pairs (e.g., about 400, about 300, about 200, about 100, about 80, about 60, about 40, about 20, about 10, or about 5 base pairs) from a target nucleic acid.

在某些實施例中，Cas 蛋白，例如 Cas9，包括功能性 RuvC 及 HNH 核酸酶域，可以裂解標靶核酸序列之兩條股。在某些實施例中，Cas 蛋白，例如 Cas9，包含一個功能性核酸內切酶域，該核酸內切酶域允許 Cas 蛋白僅裂解標靶核酸序列之一條股 (即，切口)。例如但不作為限制，Cas9 切口酶可包括：(i) 非功能性 RuvC 域 (例如，突變型 RuvC 域)，以及 (ii) 功能性 HNH 域 (例如，野生型 HNH 域)。在某些實施例中，Cas9 切口酶可包含：(i) 功能性 RuvC 域 (例如，野生型 RuvC 域)，以及 (ii) 非功能性 HNH 域 (例如，突變型 HNH 域)。在某些實施例中，Cas9 切口酶包含功能性 HNH 樣且包含 D10 處之突變，例如，D10A。在某些實施例中，Cas9 切口酶包含功能性 RuvC 域且包含 H840 處之突變，例如，H840A。在某些實施例中，Cas9 切口酶包含功能性 RuvC 域且包含 N863 處之突變，例如，N863A。In some embodiments, a Cas protein, such as Cas9, includes functional RuvC and HNH nuclease domains that can cleave both strands of a target nucleic acid sequence. In some embodiments, a Cas protein, such as Cas9, includes a functional endonuclease domain that allows the Cas protein to cleave only one strand of a target nucleic acid sequence (i.e., nick). For example, but not by way of limitation, a Cas9 nickase may include: (i) a non-functional RuvC domain (e.g., a mutant RuvC domain), and (ii) a functional HNH domain (e.g., a wild-type HNH domain). In some embodiments, a Cas9 nickase may include: (i) a functional RuvC domain (e.g., a wild-type RuvC domain), and (ii) a non-functional HNH domain (e.g., a mutant HNH domain). In some embodiments, the Cas9 nickase comprises a functional HNH-like and comprises a mutation at D10, e.g., D10A. In some embodiments, the Cas9 nickase comprises a functional RuvC domain and comprises a mutation at H840, e.g., H840A. In some embodiments, the Cas9 nickase comprises a functional RuvC domain and comprises a mutation at N863, e.g., N863A.

在某些實施例中，編碼 Cas 蛋白之核苷酸序列係密碼子最佳化的。例如但不作為限制，編碼 Cas 蛋白之核苷酸序列可以為密碼子最佳化的，例如，其中至少一個非常見的密碼子或不太常見的密碼子已被常見的密碼子替換，以在特定細胞類型 (例如，哺乳動物細胞) 中最佳化表現。In certain embodiments, the nucleotide sequence encoding the Cas protein is codon-optimized. For example, but not by way of limitation, the nucleotide sequence encoding the Cas protein can be codon-optimized, e.g., wherein at least one uncommon codon or less common codon has been replaced with a common codon for optimal expression in a particular cell type(e.g., a mammalian cell).

在某些實施例中，Cas 蛋白為包括一或多個異源蛋白域之融合蛋白。在某些實施例中，Cas 融合蛋白可以包括任何額外的蛋白域，例如，抗原決定基標籤、報導子序列及具有一或多種以下活性之蛋白域：甲基化酶活性、去甲基化酶活性、轉錄活化活性、轉錄抑制活性、轉錄釋放因子活性、組蛋白修飾活性、RNA 裂解活性及核酸結合活性。In some embodiments, the Cas protein is a fusion protein comprising one or more heterologous protein domains. In some embodiments, the Casfusion protein may include any additional protein domains, for example, an antigenic determinant tag, a reporter sequence, and a protein domain having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity.

在某些實施例中，Cas 蛋白包括一或多個核定位序列以促進 Cas 蛋白在細胞核中呈可偵測之量的累積。核定位序列為此項技術中已知的。例如但不作為限制，Cas 蛋白可以包括在其 N 端及/或 C 端處的核定位序列 (例如，來自 SV40)。IV.組成物In certain embodiments, the Cas protein includes one or more nuclear localization sequences to promote the accumulation of the Cas protein in detectable amounts in the cell nucleus. Nuclear localization sequences are known in the art. For example, but not by way of limitation, the Cas protein may include a nuclear localization sequence (e.g., from SV40) at its N-terminus and/or C-terminus.IV.Composition

本揭露進一步提供包括本文所揭示之一或多個經修飾之 gRNA 的組成物。在某些實施例中，本揭露之組成物可包括本文所揭示之兩個或更多個、三個或更多個、四個或更多個、五個或更多個、六個或更多個、七個或更多個、八個或更多個、九個或更多個、或十個或更多個經修飾之 gRNA。The present disclosure further provides compositions comprising one or more modified gRNAs disclosed herein. In certain embodiments, the compositions disclosed herein may include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more modified gRNAs disclosed herein.

在某些實施例中，本揭露之組成物包括包含本文所揭示，例如如第 II 節中所述之一或多個修飾的 gRNA。在某些實施例中，組成物可包括編碼本文所揭示之 gRNA 分子的核酸。替代地或另外，組成物包括 gRNA 分子作為經轉錄或合成的 RNA 分子。In certain embodiments, the compositions disclosed herein include a gRNA comprising one or more modifications disclosed herein, such as described in Section II. In certain embodiments, the composition may include a nucleic acid encoding a gRNA molecule disclosed herein. Alternatively or additionally, the composition includes the gRNA molecule as a transcribed or synthesized RNA molecule.

在某些實施例中，本揭露之組成物可進一步包括 RNA 引導的核酸酶。在某些實施例中，組成物包括編碼 RNA 引導的核酸酶之核酸。替代地或另外，組成物包括 RNA 引導的核酸酶作為蛋白質。在某些實施例中，RNA 引導的核酸酶為 Cas 蛋白，例如 Cas9 蛋白。In some embodiments, the compositions disclosed herein may further include an RNA-guided nuclease. In some embodiments, the composition includes a nucleic acid encoding an RNA-guided nuclease. Alternatively or additionally, the composition includes an RNA-guided nuclease as a protein. In some embodiments, the RNA-guided nuclease is a Cas protein, such as a Cas9 protein.

本揭露進一步提供包括一或多種本文所揭示之經修飾的 gRNA 之核酸組成物。在某些實施例中，核酸組成物包括編碼本文所述之 gRNA 分子的多核苷酸。在某些實施例中，核酸組成物可進一步包括編碼 RNA 引導的核酸酶 (例如 Cas 蛋白) 之多核苷酸。在某些實施例中，本揭露之核酸組成物可以包括編碼 gRNA 分子及 RNA 引導的核酸酶之核酸，例如，包括編碼 gRNA 分子之多核苷酸 (例如，偶聯至第一啟動子) 及編碼 RNA 引導的核酸酶之多核苷酸 (例如，偶聯至第二啟動子) 的核酸。在某些實施例中，本揭露之核酸組成物可包括編碼 gRNA 分子之第一核酸及編碼 RNA 引導的核酸酶之第二核酸。The present disclosure further provides a nucleic acid composition comprising one or more modified gRNAs disclosed herein. In certain embodiments, the nucleic acid composition comprises a polynucleotide encoding a gRNA molecule described herein. In certain embodiments, the nucleic acid composition may further comprise a polynucleotide encoding an RNA-guided nuclease (e.g., a Cas protein). In certain embodiments, the nucleic acid composition of the present disclosure may include a nucleic acid encoding a gRNA molecule and an RNA-guided nuclease, for example, a nucleic acid comprising a polynucleotide encoding a gRNA molecule (e.g., coupled to a first promoter) and a polynucleotide encoding an RNA-guided nuclease (e.g., coupled to a second promoter). In certain embodiments, the nucleic acid composition of the present disclosure may include a first nucleic acid encoding a gRNA molecule and a second nucleic acid encoding an RNA-guided nuclease.

在某些實施例中，編碼一或多個 gRNA 分子及/或一或多個 RNA 引導的核酸酶之核酸組成物可藉由此項技術中已知或如本文所述之方法投予至個體或遞送至細胞中。例如但不作為限制，編碼 gRNA 分子及/或 RNA 引導的核酸酶之核酸可以藉由非基於載體之方法 (例如，藉由使用 DNA 複合物或裸 DNA)、藉由基於載體之方法或其組合遞送至個體或遞送至細胞中。在某些實施例中，載體可為病毒載體。在某些實施例中，病毒為 RNA 病毒或 DNA 病毒。 例示性病毒載體/病毒包括但不限於反轉錄病毒、慢病毒、腺病毒、腺相關病毒 (AAV)、牛痘病毒、痘病毒及單純皰疹病毒。在某些實施例中，本揭露之載體包括編碼 gRNA 分子及/或 RNA 引導的核酸酶之多核苷酸序列。在某些實施例中，載體進一步包括編碼核定位的序列，編碼核定位的序列例如在 RNA 引導的核酸酶之 N 端及/或 C 端處與 RNA 引導的核酸酶融合。In certain embodiments, nucleic acid compositions encoding one or more gRNA molecules and/or one or more RNA-guided nucleases can be administered to an individual or delivered to a cell by methods known in the art or as described herein. For example, but not by way of limitation, nucleic acids encoding gRNA molecules and/or RNA-guided nucleases can be delivered to an individual or delivered to a cell by non-vector-based methods (e.g., by using DNA complexes or naked DNA), by vector-based methods, or a combination thereof. In certain embodiments, the vector can be a viral vector. In certain embodiments, the virus is an RNA virus or a DNA virus. Exemplary viral vectors/viruses include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses. In certain embodiments, the vector disclosed herein comprises a polynucleotide sequence encoding a gRNA molecule and/or an RNA-guided nuclease. In certain embodiments, the vector further comprises a sequence encoding a nuclear localization, wherein the sequence encoding a nuclear localization is fused to the RNA-guided nuclease at the N-terminus and/or C-terminus of the RNA-guided nuclease, for example.

本揭露進一步提供包含核糖核酸蛋白 (RNP) 複合物之組成物。在某些實施例中，RNP 複合物包括 gRNA 分子 (例如，作為經轉錄或合成之 RNA) 及 RNA 引導的核酸酶。 在某些實施例中，gRNA 分子在遞送至標靶細胞之前在適合的條件下與 RNA 引導的核酸酶形成 RNP 複合物。The present disclosure further provides compositions comprising ribonucleoprotein (RNP) complexes. In certain embodiments, the RNP complexes include a gRNA molecule (e.g., as transcribed or synthesized RNA) and an RNA-guided nuclease. In certain embodiments, the gRNA molecule forms an RNP complex with the RNA-guided nuclease under suitable conditions prior to delivery to a target cell.

本揭露進一步提供包括本揭露之一或多種組成物的細胞。例如但不作為限制，細胞可包括本揭露之核酸組成物。在某些實施例中，細胞可包括本揭露之 RNP 複合物。可使用所揭示之組成物來修飾多種細胞。例如但不作為限制，細胞可為免疫細胞，例如，T 細胞。在某些實施例中，T 細胞可為 CD8⁺T 細胞及/或 CD4⁺T 細胞。在某些實施例中，細胞可為幹細胞。在某些實施例中，細胞可為誘導性富潛能幹 (iPS) 細胞。在某些實施例中，細胞可為源自幹細胞或 iPS 之細胞，例如，源自幹細胞或 iPS 之免疫細胞。The present disclosure further provides cells comprising one or more compositions of the present disclosure. For example, but not by way of limitation, the cells may comprise the nucleic acid compositions of the present disclosure. In certain embodiments, the cells may comprise the RNP complexes of the present disclosure. The disclosed compositions may be used to modify a variety of cells. For example, but not by way of limitation, the cells may be immune cells, such as T cells. In certain embodiments, the T cells may be CD8⁺ T cells and/or CD4⁺ T cells. In certain embodiments, the cells may be stem cells. In certain embodiments, the cells may be induced enriched-potential stem (iPS) cells. In certain embodiments, the cell may be a stem cell-derived or iPS-derived cell, for example, a stem cell-derived or iPS-derived immune cell.

在某些實施例中，本揭露之組成物，例如，本揭露之核酸組成物，可藉由此項技術中已知或如本文所述之方法遞送至標靶細胞中。例如但不作為限制，本揭露之組成物可藉由顯微注射、電穿孔、瞬態細胞壓縮或擠壓、脂質介導之轉染、肽介導之遞送或其組合遞送至細胞中。在某些實施例中，組成物例如包含 RNP 複合物，藉由電穿孔遞送至標靶細胞。V.使用方法In certain embodiments, the compositions of the present disclosure, for example, nucleic acid compositions of the present disclosure, can be delivered to target cells by methods known in the art or as described herein. For example, but not by way of limitation, the compositions of the present disclosure can be delivered to cells by microinjection, electroporation, transient cell compression or extrusion, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. In certain embodiments, the composition, for example, comprises an RNP complex, which is delivered to a target cell by electroporation.V.Methods of Use

本揭露進一步提供使用本文所揭示之 gRNA 的方法。在某些實施例中，本文提供之 gRNA 分子可用於修飾細胞中之標靶核酸。例如但不作為限制，本揭露之 gRNA 分子可用於例如離體或活體內修飾細胞中之標靶核酸。The present disclosure further provides methods for using the gRNA disclosed herein. In certain embodiments, the gRNA molecules provided herein can be used to modify a target nucleic acid in a cell. For example, but not by way of limitation, the gRNA molecules disclosed herein can be used to modify a target nucleic acid in a cell, for example, in vitro or in vivo.

在某些實施例中，本揭露之 gRNA 分子可用於減少細胞中標靶核酸之表現的方法中。在某些實施例中，本揭露之 gRNA 分子可用於增加細胞中標靶核酸之表現的方法中。在某些實施例中，本揭露之 gRNA 分子可用於在緊鄰標靶核酸處引入一或多個核苷酸之插入或缺失的方法中。在某些實施例中，本揭露之 gRNA 分子可用於在緊鄰標靶核酸處引入缺失的方法中。在某些實施例中，本揭露之 gRNA 分子可用於在緊鄰標靶核酸處引入插入的方法中。在某些實施例中，本揭露之 gRNA 分子可用於在緊鄰標靶核酸處引入一或多個斷裂 (例如，雙股斷裂或單股斷裂) 的方法中。In certain embodiments, the gRNA molecules disclosed herein can be used in methods for reducing the expression of a target nucleic acid in a cell. In certain embodiments, the gRNA molecules disclosed herein can be used in methods for increasing the expression of a target nucleic acid in a cell. In certain embodiments, the gRNA molecules disclosed herein can be used in methods for introducing insertions or deletions of one or more nucleotides in close proximity to a target nucleic acid. In certain embodiments, the gRNA molecules disclosed herein can be used in methods for introducing deletions in close proximity to a target nucleic acid. In certain embodiments, the gRNA molecules disclosed herein can be used in methods for introducing insertions in close proximity to a target nucleic acid. In certain embodiments, the gRNA molecules disclosed herein can be used in methods for introducing one or more breaks (e.g., double-strand breaks or single-strand breaks) in close proximity to a target nucleic acid.

在某些實施例中，用於修飾細胞中之標靶核酸的方法包括使細胞與 gRNA 分子接觸，該 gRNA 分子包括對標靶核酸中之序列具有特異性的間隔子 (例如，靶向域)。在某些實施例中，該方法包括使細胞與本文所揭示之組成物 (例如，包含經修飾的 gRNA 分子及 RNA 引導的核酸酶之組成物) 接觸。In certain embodiments, a method for modifying a target nucleic acid in a cell comprises contacting the cell with a gRNA molecule comprising a spacer (e.g., a targeting domain) specific for a sequence in the target nucleic acid. In certain embodiments, the method comprises contacting the cell with a composition disclosed herein (e.g., a composition comprising a modified gRNA molecule and an RNA-guided nuclease).

本揭露進一步提供用於治療個體之方法。例如但不作為限制，包含本揭露之 gRNA 分子的組成物可用於治療方法中。在某些實施例中，本揭露提供用於治療有需要之個體的 gRNA 分子。在某些實施例中，本揭露提供用於治療患有疾病之個體的 gRNA 分子。在某些實施例中，gRNA 分子包含與標靶核酸 (例如，與待治療之疾病相關的基因) 互補的間隔子。在某些實施例中，該方法包括藉由使細胞與包含有效量之本文所揭示之 gRNA 分子或本文所揭示之 RNP 複合物的組成物接觸 (例如，藉由本文所揭示或此項技術中已知之任何遞送方法進行) 來離體修飾個體之細胞。在某些實施例中，該方法可進一步包括將經修飾的細胞送回至個體。The present disclosure further provides methods for treating an individual. For example, but not by way of limitation, a composition comprising a gRNA molecule disclosed herein can be used in a method of treatment. In certain embodiments, the present disclosure provides a gRNA molecule for treating an individual in need thereof. In certain embodiments, the present disclosure provides a gRNA molecule for treating an individual suffering from a disease. In certain embodiments, the gRNA molecule comprises a spacer that is complementary to a target nucleic acid (e.g., a gene associated with the disease to be treated). In certain embodiments, the method comprises ex vivo modifying a cell of an individual by contacting the cell with a composition comprising an effective amount of a gRNA molecule disclosed herein or an RNP complex disclosed herein (e.g., by any delivery method disclosed herein or known in the art). In certain embodiments, the method may further comprise returning the modified cells to the individual.

在某些實施例中，本揭露之 gRNA 分子可用作藥物。在某些實施例中，本揭露提供本文所揭示之 gRNA 分子在藥物之製造或製備中的用途。在某些實施例中，該藥物用於治療疾病。在某些實施例中，該藥物用於治療疾病之方法中，該方法包括向患有疾病之個體投予有效量之藥物。In certain embodiments, the gRNA molecules disclosed herein can be used as drugs. In certain embodiments, the disclosure provides the use of the gRNA molecules disclosed herein in the manufacture or preparation of drugs. In certain embodiments, the drug is used to treat a disease. In certain embodiments, the drug is used in a method of treating a disease, the method comprising administering an effective amount of the drug to an individual suffering from the disease.

在某些實施例中，包含本文提供之 gRNA 分子中之任一者的組成物例如可用於上述治療方法中之任一者中。在某些實施例中，包含本文提供之任何 gRNA 分子及/或 RNA 引導的核酸酶之組成物可用於治療方法 (例如，離體修飾細胞) 中。In certain embodiments, a composition comprising any of the gRNA molecules provided herein can be used, for example, in any of the above-described treatment methods. In certain embodiments, a composition comprising any of the gRNA molecules and/or RNA-guided nucleases provided herein can be used in a treatment method (e.g., ex vivo modification of cells).

在某些實施例中，本文所揭示之一或多種組成物，例如，核酸組成物，可以投予至個體或與來自個體之細胞接觸，例如，離體接觸。在某些實施例中，組成物可包括本揭露之 gRNA 及 RNA 引導的核酸酶。替代地，組成物包括本揭露之 gRNA 且不包括 RNA 引導的核酸酶。在某些實施例中，組成物，例如，包括 gRNA 之核酸組成物，與包括 RNA 引導的核酸酶之組成物同時投予。在某些實施例中，組成物，例如，包括 gRNA 之核酸組成物，在包括 RNA 引導的核酸酶之組成物之後投予。在某些實施例中，組成物，例如，包括 gRNA 之核酸組成物，在包括 RNA 引導的核酸酶之組成物之前投予。在某些實施例中，核酸組成物可藉由本文所述之任何遞送方法來遞送，例如，編碼 gRNA 之核酸可以藉由病毒載體遞送。在某些實施例中，包括 RNA 引導的核酸酶之組成物可以藉由電穿孔遞送至細胞。VI.基因編輯系統In certain embodiments, one or more compositions disclosed herein, e.g., nucleic acid compositions, can be administered to an individual or contacted with cells from an individual, e.g.,ex vivo . In certain embodiments, the composition may include a gRNA and an RNA-guided nuclease disclosed herein. Alternatively, the composition includes a gRNA disclosed herein and does not include an RNA-guided nuclease. In certain embodiments, a composition, e.g., a nucleic acid composition including a gRNA, is administered simultaneously with a composition including an RNA-guided nuclease. In certain embodiments, a composition, e.g., a nucleic acid composition including a gRNA, is administered after a composition including an RNA-guided nuclease. In certain embodiments, a composition, e.g., a nucleic acid composition including a gRNA, is administered before a composition including an RNA-guided nuclease. In certain embodiments, nucleic acid compositions can be delivered by any of the delivery methods described herein, for example, nucleic acids encoding gRNA can be delivered by viral vectors. In certain embodiments, compositions comprising RNA-guided nucleases can be delivered to cells by electroporation.VI.Gene Editing Systems

本揭露亦提供基因編輯系統，其包含本文所揭示之一或多種組成物及/或可用於執行本文所述之方法的材料。The present disclosure also provides gene editing systems comprising one or more compositions disclosed herein and/or materials that can be used to perform the methods described herein.

在某些實施例中，本揭露之基因編輯系統可以包含本文所揭示之一或多個經修飾之 gRNA 分子。例如但不作為限制，本揭露之基因編輯系統可以包含第 II 節中所揭示之一或多個 gRNA 分子。In certain embodiments, the gene editing system disclosed herein may include one or more modified gRNA molecules disclosed herein. For example, but not by way of limitation, the gene editing system disclosed herein may include one or more gRNA molecules disclosed in Section II.

在某些實施例中，本揭露之基因編輯系統之 gRNA 分子包含：(a) 在引導 RNA 分子的 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結；(b) 在引導 RNA 分子的 3' 端 (第「n」個) 核苷酸與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結；或 (c) 在引導 RNA 分子的 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結以及在引導 RNA 分子的第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。在某些實施例中，二硫代磷酸酯鍵結存在於 gRNA 分子的 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、以及第三個與第四個核苷酸之間。在某些實施例中，二硫代磷酸酯鍵結存在於 gRNA 分子的第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸之間、或第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間。In certain embodiments, the gRNA molecule of the gene editing system of the present disclosure comprises: (a) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule; (b) a phosphorodithioate bond between the 3' ("n") nucleotide and the n-1 nucleotide of the guide RNA molecule; or (c) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule and a phosphorodithioate bond between the nth and n-1 nucleotides of the guide RNA molecule. In certain embodiments, the phosphorodithioate bonds are present between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the gRNA molecule. In certain embodiments, phosphorodithioate bonds exist between the nth and n-1th nucleotides and the n-1th and n-2th nucleotides, or between the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the gRNA molecule.

在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個及/或第三個核苷酸包含選自由以下所組成之群組的修飾：經 2'-氟修飾的核苷酸；經 2'-O-甲基修飾的核苷酸；經 2'-O-(2-甲氧基乙基) 修飾的核苷酸；鎖核酸 (LNA)；去氧核糖核苷酸；以及上述修飾中之兩者或更多者的組合。在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個、第三個及/或第四個核苷酸包含選自由以下所組成之群組的修飾：經 2'-氟修飾的核苷酸；經 2'-O-甲基修飾的核苷酸；經 2'-O-(2-甲氧基乙基) 修飾的核苷酸；鎖核酸 (LNA)；去氧核糖核苷酸；以及上述修飾中之兩者或更多者的組合。在某些實施例中，gRNA 分子的 5' 端處的第一個、第二個、第三個、第四個及/或第五個核苷酸包含選自由以下所組成之群組的修飾：經 2'-氟修飾的核苷酸；經 2'-O-甲基修飾的核苷酸；經 2'-O-(2-甲氧基乙基) 修飾的核苷酸；鎖核酸 (LNA)；去氧核糖核苷酸；以及上述修飾中之兩者或更多者的組合。In certain embodiments, the first, second, and/or third nucleotide at the 5' end of the gRNA molecule comprises a modification selected from the group consisting of: a 2'-fluoro modified nucleotide; a 2'-O-methyl modified nucleotide; a 2'-O-(2-methoxyethyl) modified nucleotide; a locked nucleic acid (LNA); a deoxyribonucleotide; and a combination of two or more of the above modifications. In certain embodiments, the first, second, third, and/or fourth nucleotide at the 5' end of the gRNA molecule comprises a modification selected from the group consisting of: a 2'-fluoro modified nucleotide; a 2'-O-methyl modified nucleotide; a 2'-O-(2-methoxyethyl) modified nucleotide; a locked nucleic acid (LNA); a deoxyribonucleotide; and a combination of two or more of the above modifications. In certain embodiments, the first, second, third, fourth, and/or fifth nucleotide at the 5' end of the gRNA molecule comprises a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a locked nucleic acid (LNA); a deoxyribonucleotide; and a combination of two or more of the foregoing modifications.

在某些實施例中，gRNA 分子之第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個、第 n-1 個與第 n-2 個或第 n 個、第 n-1 個與第 n-2 個核苷酸包含選自由以下所組成之群組的修飾：經 2'-氟修飾的核苷酸；經 2'-O-甲基修飾的核苷酸；經 2'-O-(2-甲氧基乙基) 修飾的核苷酸；鎖核酸 (LNA)；去氧核糖核苷酸；以及上述修飾中之兩者或更多者的組合。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸或第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸包含選自由以下所組成之群組的修飾：經 2'-氟修飾的核苷酸；經 2'-O-甲基修飾的核苷酸；經 2'-O-(2-甲氧基乙基) 修飾的核苷酸；鎖核酸 (LNA)；去氧核糖核苷酸；以及上述修飾中之兩者或更多者的組合。在某些實施例中，gRNA 分子之第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸包含選自由以下所組成之群組的修飾：經 2'-氟修飾的核苷酸；經 2'-O-甲基修飾的核苷酸；經 2'-O-(2-甲氧基乙基) 修飾的核苷酸；鎖核酸 (LNA)；去氧核糖核苷酸；以及上述修飾中之兩者或更多者的組合。In certain embodiments, the nth and n-1th nucleotides, the nth and n-2th, the n-1th and n-2th, or the nth, n-1th and n-2th nucleotides of the gRNA molecule comprise a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a locked nucleic acid (LNA); a deoxyribonucleotide; and a combination of two or more of the foregoing modifications. In certain embodiments, the nth, n-1th, n-2th, and n-3th nucleotides or the n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule comprise a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a locked nucleic acid (LNA); a deoxyribonucleotide; and a combination of two or more of the foregoing modifications. In certain embodiments, the nth, n-1th, n-2th, n-3th, and n-4th nucleotides of the gRNA molecule comprise a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a locked nucleic acid (LNA); a deoxyribonucleotide; and a combination of two or more of the foregoing modifications.

在某些實施例中，本揭露之基因編輯系統可包括一或多個經修飾的 gRNA (例如如第 II 節中所揭示)，及 RNA 引導的核酸酶 (例如如第 III 節中所揭示之 RNA 引導的核酸酶)。在某些實施例中，RNA 引導的核酸酶為 Cas 蛋白。在某些實施例中，Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。In some embodiments, the gene editing system disclosed herein may include one or more modified gRNAs(e.g., as disclosed in Section II), and RNA-guided nucleases(e.g., RNA-guided nucleases disclosed in Section III). In some embodiments, the RNA-guided nuclease is a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of: Cas9, Cas12, Cas13, and combinations thereof.

在某些實施例中，本揭露之基因編輯系統包含編碼如本文所述，(例如如第 II 節中所揭示) 之 gRNA 的一或多種核酸。在某些實施例中，一或多種核酸進一步包含編碼 RNA 引導的核酸酶 (例如如第 III 節中所揭示之 RNA 引導的核酸酶) 的多核苷酸。在某些實施例中，RNA 引導的核酸酶為 Cas 蛋白。在某些實施例中，Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。In some embodiments, the gene editing system disclosed herein comprises one or more nucleic acids encoding a gRNA as described herein (e.g., as disclosed in Section II). In some embodiments, the one or more nucleic acids further comprise a polynucleotide encoding an RNA-guided nuclease (e.g., an RNA-guided nuclease as disclosed in Section III). In some embodiments, the RNA-guided nuclease is a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of: Cas9, Cas12, Cas13, and combinations thereof.

在某些實施例中，基因編輯系統包含一或多種載體，該一或多種載體包含如本文所述，例如如第 IV 節中所揭示之核酸。In certain embodiments, the gene editing system comprises one or more vectors comprising a nucleic acid as described herein, e.g., as disclosed in Section IV.

在某些實施例中，基因編輯系統包含一或多種組成物，該一或多種組成物包含本文所揭示，例如如第 II 節中所揭示之 gRNA。在某些實施例中，組成物進一步包含 RNA 引導的核酸酶。在某些實施例中，組成物包含本文所述，例如如第 IV 節中所揭示之核酸。In some embodiments, the gene editing system comprises one or more compositions comprising a gRNA disclosed herein, such as disclosed in Section II. In some embodiments, the composition further comprises an RNA-guided nuclease. In some embodiments, the composition comprises a nucleic acid described herein, such as disclosed in Section IV.

在某些實施例中，基因編輯系統包含一或多種 RNP 複合物，該一或多種 RNP 複合物包含第 II 節及本文所揭示之 gRNA 以及 RNA 引導的核酸酶。在某些實施例中，RNA 引導的核酸酶為 Cas 蛋白。在某些實施例中，Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。VII.示例性實施例In some embodiments, the gene editing system comprises one or more RNP complexes, which comprise the gRNA disclosed in Section II and herein and an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of: Cas9, Cas12, Cas13, and combinations thereof.VII.Exemplary Embodiments

A1.   本發明所揭示之標的提供一種引導 RNA 分子，其包含： (a) 在該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結； (b) 在該引導 RNA 分子的 3' 端 (第「n」個) 核苷酸與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結；或 (c) 在該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結以及在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。A1.   The subject matter disclosed in the present invention provides a guide RNA molecule comprising:(a) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule;(b) a phosphorodithioate bond between the 3' end ("n"th) nucleotide and the n-1th nucleotide of the guide RNA molecule; or(c) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule and a phosphorodithioate bond between the nth and n-1th nucleotides of the guide RNA molecule.

A2.  如 A1 之引導 RNA 分子，其進一步包含在該引導 RNA 分子的 5' 端處的第二個與第三個核苷酸以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。A2. A guide RNA molecule as in A1, further comprising phosphorodithioate bonds between the second and third nucleotides and the third and fourth nucleotides at the 5' end of the guide RNA molecule.

A3.  如 A1 或 A2 之引導 RNA 分子，其包含在該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。A3. A guide RNA molecule as A1 or A2, comprising phosphorodithioate bonds between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the guide RNA molecule.

A4.  如 A1 至 A3 中任一項之引導 RNA 分子，其包含在該引導 RNA 分子的 5' 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、第三個與第四個核苷酸、以及第四個與第五個核苷酸之間的二硫代磷酸酯鍵結。A4. A guide RNA molecule as in any one of A1 to A3, comprising dithiophosphate bonds between the first and second nucleotides, the second and third nucleotides, the third and fourth nucleotides, and the fourth and fifth nucleotides at the 5' end of the guide RNA molecule.

A5.   如 A1 至 A4 中任一項之引導 RNA 分子，其包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸或者第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。A5. A guide RNA molecule as in any one of A1 to A4, comprising a phosphorothioate bond between the nth and n-1th nucleotides and the n-1th and n-2th nucleotides or between the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the guide RNA molecule.

A6.   如 A1 至 A5 中任一項之引導 RNA 分子，其包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸、第 n-1 個與第 n-2 個核苷酸、以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。A6. A guide RNA molecule as in any one of A1 to A5, comprising dithiophosphate bonds between the nth and n-1th nucleotides, the n-1th and n-2th nucleotides, and the n-2th and n-3th nucleotides of the guide RNA molecule.

A7.  如 A1 至 A6 中任一項之引導 RNA 分子，其包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸、第 n-1 個與第 n-2 個核苷酸、第 n-2 個與第 n-3 個核苷酸、以及第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。A7. A guide RNA molecule as in any one of A1 to A6, comprising dithiophosphate bonds between the nth and n-1th nucleotides, the n-1th and n-2th nucleotides, the n-2th and n-3th nucleotides, and the n-3th and n-4th nucleotides of the guide RNA molecule.

A8.   如 A1 至 A7 中任一項之引導 RNA 分子，其中在該引導 RNA 分子的 5' 端處的第一個核苷酸包含選自由以下所組成之群組的修飾： (a) 經 2’-氟修飾的核苷酸； (b) 經 2’-O-甲基修飾的核苷酸； (c) 經 2’-O-(2-甲氧基乙基) 修飾的核苷酸； (d) 鎖核酸 (LNA)； (e) 去氧核糖核苷酸；以及 (f)  (a) 至 (e) 中之兩者或更多者之組合。A8. A guide RNA molecule as in any one of A1 to A7, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a modification selected from the group consisting of:(a) 2'-fluorine-modified nucleotides;(b) 2'-O-methyl-modified nucleotides;(c) 2'-O-(2-methoxyethyl)-modified nucleotides;(d) locked nucleic acids (LNA);(e) deoxyribonucleotides; and(f) a combination of two or more of (a) to (e).

A9.   如 A8 之引導 RNA 分子，其中在該引導 RNA 分子的 5' 端處的第一個核苷酸包含經 2'-氟修飾的核苷酸。A9. A guide RNA molecule as in A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-fluorine-modified nucleotide.

A10. 如 A8 之引導 RNA 分子，其中在該引導 RNA 分子的 5' 端處的第一個核苷酸包含經 2'-O-甲基修飾的核苷酸。A10. The guide RNA molecule of A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-O-methyl modified nucleotide.

A11. 如 A8 之引導 RNA 分子，其中在該引導 RNA 分子的 5' 端處的第一個核苷酸包含經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。A11. The guide RNA molecule of A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl)-modified nucleotide.

A12. 如 A8 之引導 RNA 分子，其中在該引導 RNA 分子的 5' 端處的第一個核苷酸包含 LNA。A12. The guide RNA molecule of A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises LNA.

A13. 如 A8 之引導 RNA 分子，其中在該引導 RNA 分子的 5' 端處的第一個核苷酸包含去氧核糖核苷酸。A13. The guide RNA molecule of A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a deoxyribonucleotide.

A14. 如 A1 至 A13 中任一項之引導 RNA 分子，其中該引導 RNA 分子的 5' 端處的 (i) 第二個核苷酸、(ii) 第三個核苷酸、(iii) 第四個核苷酸、(iv) 第二個與第三個核苷酸、(v) 第二個與第四個核苷酸、(vi) 第三個與第四個核苷酸或 (v) 第二個、第三個與第四個核苷酸包含選自由以下所組成之群組的修飾： (a) 經 2’-氟修飾的核苷酸； (b) 經 2’-O-甲基修飾的核苷酸； (c) 經 2’-O-(2-甲氧基乙基) 修飾的核苷酸； (d) 鎖核酸 (LNA)； (e) 去氧核糖核苷酸；以及 (f)  (a) 至 (e) 中之兩者或更多者之組合。A14. A guide RNA molecule as described in any one of A1 to A13, wherein (i) the second nucleotide, (ii) the third nucleotide, (iii) the fourth nucleotide, (iv) the second and third nucleotides, (v) the second and fourth nucleotides, (vi) the third and fourth nucleotides, or (v) the second, third and fourth nucleotides at the 5' end of the guide RNA molecule comprise a modification selected from the group consisting of: (a) 2'-fluorine-modified nucleotides; (b) 2'-O-methyl-modified nucleotides; (c) 2'-O-(2-methoxyethyl)-modified nucleotides; (d) locked nucleic acids (LNA); (e) deoxyribonucleotides; and (f) a combination of two or more of (a) to (e).

A15. 如 A1 至 A9 及 A14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包含三個連續的經 2'-氟修飾的核苷酸。A15. The guide RNA molecule of any one of A1 to A9 and A14, wherein the guide RNA molecule comprises three consecutive 2'-fluorine-modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.

A16. 如 A1 至 A8、A10 及 A14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包含三個連續的經 2'-O-甲基修飾的核苷酸。A16. The guide RNA molecule of any one of A1 to A8, A10 and A14, wherein the guide RNA molecule comprises three consecutive 2'-O-methyl-modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.

A17. 如 A1 至 A8、A10 及 A14 中任一項之引導 RNA 分子，其中該引導 RNA 分子於該引導 RNA 分子的 5' 端處的前四個核苷酸處包含四個連續的經 2'-O-甲基修飾的核苷酸。A17. The guide RNA molecule of any one of A1 to A8, A10 and A14, wherein the guide RNA molecule comprises four consecutive 2'-O-methyl-modified nucleotides at the first four nucleotides at the 5' end of the guide RNA molecule.

A18. 如 A1 至 A8、A11 及 A14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包含三個連續的經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。A18. The guide RNA molecule of any one of A1 to A8, A11 and A14, wherein the guide RNA molecule comprises three consecutive 2'-O-(2-methoxyethyl)-modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.

A19. 如 A1 至 A8、A12 及 A14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包含三個連續的 LNA。A19. The guide RNA molecule of any one of A1 to A8, A12 and A14, wherein the guide RNA molecule comprises three consecutive LNAs at the first three nucleotides at the 5' end of the guide RNA molecule.

A20. 如 A1 至 A8、A13 及 A14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5' 端處的前三個核苷酸處包含三個連續的去氧核糖核苷酸。A20. The guide RNA molecule of any one of A1 to A8, A13 and A14, wherein the guide RNA molecule comprises three consecutive deoxyribonucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.

A21. 如 A1 至 A20 中任一項之引導 RNA 分子，其中該引導 RNA 分子的 3' 端 (第「n」個) 核苷酸包含選自由以下所組成之群組的修飾： (a) 經 2'-氟修飾的核苷酸； (b) 經 2'-O-甲基修飾的核苷酸； (c) 經 2'-O-(2-甲氧基乙基) 修飾的核苷酸； (d) 鎖核酸 (LNA)； (e) 去氧核糖核苷酸；以及 (f) (a) 至 (e) 中之兩者或更多者之組合。A21. A guide RNA molecule as described in any one of A1 to A20, wherein the 3'-end ("n"-th) nucleotide of the guide RNA molecule comprises a modification selected from the group consisting of:(a) 2'-fluorine-modified nucleotides;(b) 2'-O-methyl-modified nucleotides;(c) 2'-O-(2-methoxyethyl)-modified nucleotides;(d) locked nucleic acids (LNA);(e) deoxyribonucleotides; and(f) a combination of two or more of (a) to (e).

A22. 如 A21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含經 2'-氟修飾的核苷酸。A22. The guide RNA molecule of A21, wherein the nth nucleotide of the guide RNA molecule comprises a 2'-fluorine-modified nucleotide.

A23. 如 A21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含經 2'-O-甲基修飾的核苷酸。A23. The guide RNA molecule of A21, wherein the nth nucleotide of the guide RNA molecule comprises a 2'-O-methyl modified nucleotide.

A24. 如 A21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。A24. The guide RNA molecule of A21, wherein the nth nucleotide of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl)-modified nucleotide.

A25. 如 A21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含 LNA。A25. The guide RNA molecule of A21, wherein the nth nucleotide of the guide RNA molecule comprises LNA.

A26. 如 A21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含去氧核糖核苷酸。A26. The guide RNA molecule of A21, wherein the nth nucleotide of the guide RNA molecule comprises a deoxyribonucleotide.

A27. 如 A1 至 A26 中任一項之引導 RNA 分子，其中該引導 RNA 分子的 (i) 第 n-1 個核苷酸、(ii) 第 n-2 個核苷酸、(iii) 第 n-3 個核苷酸、(iv) 第 n-4 個核苷酸、(v) 第 n 個與第 n-1 個核苷酸、(vi) 第 n 個與第 n-2 個核苷酸、(vii) 第 n-1 個與第 n-2 個核苷酸、(viii) 第 n 個、第 n-1 個與第 n-2 個核苷酸、(ix) 第 n-1 個、第 n-2 個與第 n-3 個核苷酸、(x) 第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸、或 (xi) 第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自包含選自由以下所組成之群組的修飾： (a) 經 2’-氟修飾的核苷酸； (b) 經 2’-O-甲基修飾的核苷酸； (c) 經 2’-O-(2-甲氧基乙基) 修飾的核苷酸； (d) 鎖核酸 (LNA)； (e) 去氧核糖核苷酸；以及 (f)  (a) 至 (e) 中之兩者或更多者之組合。A27. The guide RNA molecule of any one of A1 to A26, wherein (i) the n-1th nucleotide, (ii) the n-2th nucleotide, (iii) the n-3th nucleotide, (iv) the n-4th nucleotide, (v) the n-th and n-1th nucleotides, (vi) the n-th and n-2th nucleotides, (vii) the n-1th and n-2th nucleotides, (viii) the n-th, n-1th and n-2th nucleotides, (ix) the n-1th, n-2th and n-3th nucleotides, (x) the n-th, n-1th, n-2th and n-3th nucleotides, or (xi) the n-th, n-1th, n-2th, n-3th and n-4th nucleotides. Each of the nucleotides comprises a modification selected from the group consisting of:(a) 2’-fluorine-modified nucleotides;(b) 2’-O-methyl-modified nucleotides;(c) 2’-O-(2-methoxyethyl)-modified nucleotides;(d) locked nucleic acids (LNA);(e) deoxyribonucleotides; and(f) a combination of two or more of (a) to (e).

A28. 如 A1 至 A22 及 A27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-氟修飾的核苷酸。A28. The guide RNA molecule of any one of A1 to A22 and A27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-fluorine-modified nucleotides.

A29. 如 A1 至 A21、A23 及 A27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-O-甲基修飾的核苷酸。A29. The guide RNA molecule of any one of A1 to A21, A23 and A27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-O-methyl modified nucleotides.

A30. 如 A1 至 A21、A23、A27 及 A29 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-O-甲基修飾的核苷酸。A30. The guide RNA molecule of any one of A1 to A21, A23, A27 and A29, wherein the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-O-methyl modified nucleotides.

A31. 如 A1 至 A21、A24 及 A27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。A31. The guide RNA molecule of any one of A1 to A21, A24 and A27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-O-(2-methoxyethyl)-modified nucleotides.

A32. 如 A1 至 A21、A25 及 A27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為 LNA。A32. The guide RNA molecule of any one of A1 to A21, A25 and A27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each LNA.

A33. 如 A1 至 A21、A26 及 A27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為去氧核糖核苷酸。A33. The guide RNA molecule of any one of A1 to A21, A26 and A27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each deoxyribonucleotides.

B.     本發明所揭示之標的提供一種核酸，其包含編碼如 A1 至 A33 中任一項之引導 RNA 分子的多核苷酸。B.     The subject matter disclosed by the present invention provides a nucleic acid comprising a polynucleotide encoding a guide RNA molecule such as any one of A1 to A33.

B1.   如 B 之核酸，其進一步包含編碼 RNA 引導的核酸酶的多核苷酸。B1.   The nucleic acid as described in B, further comprising a polynucleotide encoding an RNA-guided nuclease.

B2.   如 B1 之核酸，其中該 RNA 引導的核酸酶為 Cas 蛋白。B2. A nucleic acid as in B1, wherein the RNA-guided nuclease is a Cas protein.

B3.   如 B2 之核酸，其中該 Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。B3. A nucleic acid as described in B2, wherein the Cas protein is selected from the group consisting of: Cas9, Cas12, Cas13 and combinations thereof.

C.     本發明所揭示之標的提供一種載體，其包含如 B 至 B3 中任一項之核酸。C. The subject matter disclosed in the present invention provides a vector comprising a nucleic acid as described in any one of B to B3.

D.    本發明所揭示之標的提供一種組成物，其包含如 A1 至 A33 中任一項之引導 RNA 分子。D. The subject matter disclosed in the present invention provides a composition comprising a guide RNA molecule as any one of A1 to A33.

D1.  如 D 之組成物，其進一步包含 RNA 引導的核酸酶。D1. A composition as in D, further comprising an RNA-guided nuclease.

D2.  如 D 或 D1 之組成物，其進一步包含編碼該 RNA 引導的核酸酶的核酸。D2. A composition as described in D or D1, further comprising a nucleic acid encoding the RNA-guided nuclease.

D3.  如 D1 或 D2 之組成物，其中該 RNA 引導的核酸酶為 Cas 蛋白。D3. A composition as described in D1 or D2, wherein the RNA-guided nuclease is a Cas protein.

E.     本發明所揭示之標的提供一種組成物，其包含如 B 至 B3 之核酸。E.     The subject matter disclosed in the present invention provides a composition comprising nucleic acids such as B to B3.

F.     本發明所揭示之標的提供一種組成物，其包含如 C 之載體。F. The subject matter disclosed in the present invention provides a composition comprising a carrier such as C.

G.    本發明所揭示之標的提供一種核糖核酸蛋白 (RNP) 複合物，其包含如 A1 至 A33 中任一項之引導 RNA 分子以及 RNA 引導的核酸酶。G. The subject matter disclosed in the present invention provides a ribonucleoprotein (RNP) complex comprising a guide RNA molecule such as any one of A1 to A33 and an RNA-guided nuclease.

G1.  如 G 之 RNP 複合物，其中該 RNA 引導的核酸酶為 Cas 蛋白。G1. An RNP complex as in G, wherein the RNA-guided nuclease is a Cas protein.

G2.  如 G1 之 RNP 複合物，其中該 Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。G2. An RNP complex as in G1, wherein the Cas protein is selected from the group consisting of: Cas9, Cas12, Cas13, and combinations thereof.

H.    本發明所揭示之標的提供一種細胞，其包含如 D 至 D3、E 或 F 中任一項之組成物。H. The subject matter disclosed in the present invention provides a cell comprising a composition as any one of D to D3, E or F.

I.      本發明所揭示之標的提供一種細胞，其包含如 G 至 G2 中任一項之 RNP 複合物。I. The subject matter disclosed in the present invention provides a cell comprising an RNP complex such as any one of G to G2.

J.      本發明所揭示之標的提供一種修飾細胞之方法，其包含使該細胞與如 D 至 D3、E 或 F 中任一項之組成物或如 G 至 G2 中任一項之 RNP 複合物接觸。J. The subject matter disclosed in the present invention provides a method for modifying a cell, which comprises contacting the cell with a composition such as any one of D to D3, E or F or an RNP complex such as any one of G to G2.

J1.    如 J 之方法，其中該接觸包含藉由電穿孔將該組成物引入該細胞中。J1. The method of J, wherein the contacting comprises introducing the composition into the cell by electroporation.

K.     本發明所揭示之標的提供一種治療有需要之個體之方法，其包含： (a) 藉由使該個體的細胞與如 D 至 D3、E 或 F 中任一項之組成物或如 G 至 G2 中任一項之 RNP 複合物接觸，離體修飾該細胞；以及 (b) 將經修飾的細胞送回至該個體。K.     The subject matter disclosed in the present invention provides a method for treating an individual in need thereof, comprising:(a) ex vivo modifying a cell of the individual by contacting the cell with a composition such as any one of D to D3, E or F or an RNP complex such as any one of G to G2; and(b) returning the modified cell to the individual.

L.     本發明所揭示之標的提供一種基因編輯系統，其包含： (a) 一或多個如 A1 至 A33 中任一項之引導 RNA 分子； (b) 一或多個如 B 至 B3 中任一項之核酸； (c) 一或多個如 C 之載體； (d) 一或多個如 D 至 D3、E 或 F 之組成物；及/或 (e) 一或多個如 G 至 G2 中任一項之 RNP 複合物。實例L. The subject matter disclosed in the present invention provides a gene editing system, which comprises: (a) one or more guide RNA molecules such as any one of A1 to A33; (b) one or more nucleic acids such as any one of B to B3; (c) one or more vectors such as C; (d) one or more compositions such as D to D3, E or F; and/or (e) one or more RNP complexes such as any one of G to G2.Example

藉由參考以下實例將更好地理解本發明所揭示之標的，該等實例係作為本發明所揭示之標的之例示而非作為限制而提供的。實例1：gRNA分子之修飾The subject matter disclosed in the present invention will be better understood by reference to the following examples, which are provided as illustrations of the subject matter disclosed in the present invention and not as limitations.Example1:Modification ofgRNA molecules

本實例提供經化學合成以在 gRNA 分子之 5' 端及 3' 端處包括多個修飾的 gRNA 分子。如表 1 中所示，本實例提供修飾策略 A-L。表 1 提供與參考 gRNA 分子相比，對於此等策略中之各種策略的修飾的總結。This example provides gRNA molecules that are chemically synthesized to include multiple modifications at the 5' and 3' ends of the gRNA molecules. As shown in Table 1, this example provides modification strategies A-L. Table 1 provides a summary of the modifications for each of these strategies compared to a reference gRNA molecule.

核苷酸修飾如下在表 1 中指示：*：硫代磷酸酯 (PS) 鍵結；**：二硫代磷酸酯 (PS2) 鍵結；m：2'-OMe；ln：鎖核酸 (LNA)；f：2'-氟；M：2'-O-MOE；以及 d：去氧核糖核苷酸。例如，mA 代表 2'-O-甲基腺苷，且 dA 代表腺苷去氧核糖核苷酸。表 1 之序列中引用的「N」代表任何核苷酸鹼基，例如、鳥嘌呤 (G)、腺嘌呤 (A)、胸腺嘧啶 (T) 或胞嘧啶 (C)。表 1 中所揭示之參考 gRNA 稱為參考 gRNA 1、參考 gRNA 2 及參考 gRNA 3，其具有與策略 A-L 之經修飾之 gRNA 相同的核苷酸序列。參考 gRNA 1 及參考 gRNA 3 具有相同的修飾策略，如表 1 中所示。與參考 gRNA 1 及參考 gRNA 3 相比，參考 gRNA 2 在 3' 端處具有 3 個硫代磷酸酯鍵結，且以 rU 終止。 表 1gRNA名稱5'端處之修飾策略3'端處之修飾策略序列參考 gRNA 1•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 個硫代磷酸酯鍵結•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  2 個硫代磷酸酯鍵結5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'參考 gRNA 2•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 個硫代磷酸酯鍵結•  對第 n-1 個與第 n-2 個核苷酸之 2'-O-甲基修飾 •  3 個硫代磷酸酯鍵結5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrU*mU*mU*rU-3'參考 gRNA 3•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 個硫代磷酸酯鍵結•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  2 個硫代磷酸酯鍵結5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'A•  用 LNA 替換經 2'-O-甲基修飾的核苷酸•  用 LNA 替換經 2'-O-甲基修飾的核苷酸5'-lnN*lnN*lnN*rNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUlnU*lnU*lnU-3'B•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結5'-mN**mN**mN**rNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3'C•  用經 2'-O-(2-甲氧基乙基) 修飾的核苷酸替換經 2'-O-甲基修飾的核苷酸•  用經 2'-O-(2-甲氧基乙基) 修飾的核苷酸替換經 2'-O-甲基修飾的核苷酸5'-MN*MN*MN*rNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUMU*MU*MU-3'D•  用經 2'-氟修飾的核苷酸替換經 2'-O-甲基修飾的核苷酸•  用經 2'-氟修飾的核苷酸替換經 2'-O-甲基修飾的核苷酸5'-fN*fN*fN*rNrNrNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUfU*fU*fU-3'E•  相對於參考 gRNA 1 無變化•  相對於參考 gRNA 1 無變化5'-mN*mN*mN*fNfNfNfNfNfNfNfNfNfN fNfNfNfNfNfNfNfGfUfUfUfUfAfGfAfGfCfUfAfGfAfAfAfUfAfGfCfAfAfGfUfUfAfAfAfAfUfAfAfGfGfCfUfAfGfUfCfCfGfUfUfAfUfCfAfAfCfUfUfGfAfAfAfAfAfGfUfGfGfCfAfCfCfGfAfGfUfCfGfGfUfGfCfUmU*mU*mU-3'F•  用去氧核糖核苷酸替換經 2'-O-甲基修飾的核苷酸 •  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結•  用去氧核糖核苷酸替換經 2'-O-甲基修飾的核苷酸 •  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結5'-dN**dN**dN**rNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUdU**dU**dU-3'G•  移除硫代磷酸酯鍵結•  移除硫代磷酸酯鍵結5'-mNmNmNrNrNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmUmU mU-3'H•  相對於參考 gRNA 1 無變化•  相對於參考 gRNA 1 無變化5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrNrGrU*rU*rU*rU*rA*rG*rArGrCrUrArGrArArArUrArGrCrArArG*rU*rU*rArA*rArA*rUrA*rArGrGrCrUrArG*rU*rCrCrG*rU*rU*rA*rUrCrA*rA*rCrUrUrGrArArArArArGrUrGrG*rCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'I•  相對於參考 gRNA 1 無變化•  相對於參考 gRNA 1 無變化5'-mN*mN*mN*dNdNdNdNdNdNdNdN dNdNdNdNdNdNdNdNdNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'J•  移除 2'-O-甲基修飾•  移除 2'-O-甲基修飾5'-rN*rN*rN*rNrNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrU*rU*rU-3'K•  用去氧核糖核苷酸替換經 2'-O-甲基修飾的核苷酸 •  3 二硫代磷酸酯鍵結•  相對於參考 gRNA 1 無變化5'-dN**dN**dN**rNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'L•  移除所有修飾•  相對於參考 gRNA 1 無變化5'-rNrNrNrNrNrNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'Nucleotide modifications are indicated in Table 1 as follows: *: phosphorothioate (PS) linkage; **: phosphorodithioate (PS2) linkage; m: 2'-OMe; ln: locked nucleic acid (LNA); f: 2'-fluoro; M: 2'-O-MOE; and d: deoxyribonucleotide. For example, mA represents 2'-O-methyladenosine, and dA represents adenosine deoxyribonucleotide. "N" cited in the sequences of Table 1 represents any nucleotide base, for example, guanine (G), adenine (A), thymine (T) or cytosine (C). The reference gRNAs disclosed in Table 1 are referred to as reference gRNA 1, reference gRNA 2, and reference gRNA 3, which have the same nucleotide sequence as the modified gRNA of Strategy AL. Reference gRNA 1 and reference gRNA 3 have the same modification strategy, as shown in Table 1. Compared with reference gRNA 1 and reference gRNA 3, reference gRNA 2 has 3 phosphorothioate bonds at the 3' end and terminates with rU. Table 1gRNAName5'modification strategyModification strategies at the3' endsequence Reference gRNA 1 • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorothioate bonds • 2'-O-methyl modification of the terminal 3 nucleotides • 2 phosphorothioate bonds 5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrAr GrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3' Reference gRNA 2 • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorothioate bonds • 2'-O-methyl modification of the n-1 and n-2 nucleotides • 3 phosphorothioate bonds 5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArG rUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrU*mU*mU*rU-3' Reference gRNA 3 • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorothioate bonds • 2'-O-methyl modification of the terminal 3 nucleotides • 2 phosphorothioate bonds 5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrAr GrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3' A • Replacement of 2'-O-methyl modified nucleotides with LNA • Replacement of 2'-O-methyl modified nucleotides with LNA 5'-lnN*lnN*lnN*rNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArG rUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUlnU*lnU*lnU-3' B • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replacement of phosphorothioate bonds with phosphorodithioate bonds 5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrNrNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArUrArArGrGrCrUrAr GrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3' C • Replacement of 2'-O-methyl modified nucleotides with 2'-O-(2-methoxyethyl) modified nucleotides • Replacement of 2'-O-methyl modified nucleotides with 2'-O-(2-methoxyethyl) modified nucleotides 5'-MN*MN*MN*rNrNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrAr GrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUMU*MU*MU-3' D • Replacement of 2'-O-methyl modified nucleotides with 2'-fluoro modified nucleotides • Replacement of 2'-O-methyl modified nucleotides with 2'-fluoro modified nucleotides 5'-fN*fN*fN*rNrNrNrNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArUrArArGrGrCrUrArGr UrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUfU*fU*fU-3' E • No change relative to reference gRNA 1 • No change relative to reference gRNA 1 5'-mN*mN*mN*fNfNfNfNfNfNfNfNfNfN fNfNfNfNfNfNfNfGfUfUfUfUfAfGfAfGfCfUfAfGfAfAfAfUfAfGfCfAfAfGfUfUfAfAfAfAfUfAfAfGfGfCfUfAf GfUfCfCfGfUfUfAfUfCfAfAfCfUfUfGfAfAfAfAfGfUfGfGfCfAfCfCfGfAfGfUfCfGfGfUfGfCfUmU*mU*mU-3' F • Replacement of 2'-O-methyl modified nucleotides with deoxyribonucleotides • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replacement of 2'-O-methyl modified nucleotides with deoxyribonucleotides • Replacement of phosphorothioate bonds with phosphorodithioate bonds 5'-dN**dN**dN**rNrNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArG rUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUdU**dU**dU-3' G • Removal of phosphorothioate bonds • Removal of phosphorothioate bonds 5'-mNmNmNrNrNrNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrU rArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmUmU mU-3' H • No change relative to reference gRNA 1 • No change relative to reference gRNA 1 5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrNr NrNrNrNrNrNrNrNrGrU*rU*rU*rU*rA*rG*rArGrCrUrArGrArArArUrArGrCrArArG*rU*rU*rArA*rArA*rUrA*rArGrGrCrUr ArG*rU*rCrCrG*rU*rU*rA*rUrCrA*rA*rCrUrUrGrArArArArGrUrGrG*rCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3' I • No change relative to reference gRNA 1 • No change relative to reference gRNA 1 5'-mN*mN*mN*dNdNdNdNdNdNdNdN dNdNdNdNdNdNdNdNdNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArUrArArGrGrCrUr ArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3' J • Removal of 2'-O-methyl modification • Removal of 2'-O-methyl modification 5'-rN*rN*rN*rNrNrNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArG rUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrU*rU*rU-3' K • Replacement of 2'-O-methyl modified nucleotides with deoxyribonucleotides • 3 Phosphorodithioate bonds • No change relative to reference gRNA 1 5'-dN**dN**dN**rNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrNrNrNrGrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrA rGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3' L • Remove all modifications • No change relative to reference gRNA 1 5'-rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrN rNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArUrArArGrGrCrUrArGr UrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'

合成的經修飾之 gRNA 的身份藉由質譜法來確認，實驗質量與理論質量之間的質量誤差小於 200 ppm。如表 2 及表 3 中所示，實驗質量與計算質量一致，證實了長度及核鹼基組成為正確的。The identity of the synthesized modified gRNA was confirmed by mass spectrometry, and the mass error between the experimental and theoretical masses was less than 200 ppm. As shown in Tables 2 and 3, the experimental masses were consistent with the calculated masses, confirming that the length and base composition were correct.

對合成的經修飾之 gRNA 的純度進行分析。如圖2 及圖 3 中所示，對於根據修飾方案 A-F 進行修飾之 gRNA，觀察到相似的純度概況。此等經修飾之 gRNA 的純度範圍在 80.1% 與 99.5% 之間。圖2 及圖 4 以及表 2 提供了靶向參考標靶 1 且根據修飾方案 A-L 進行修飾之 gRNA 的純度概況，且圖3 及圖 5 以及表 3 提供了靶向參考標靶 2 且根據修飾方案 A-L 進行修飾之 gRNA 的純度概況。如圖2 及圖 3 中所示，gRNA 之序列對具有根據修飾方案 A-F 之修飾的經修飾之 gRNA 的純度及離子對逆相液相層析 (IPRP) 概況具有可忽略的影響。對於靶向此兩個標靶序列且具有根據修飾方案 G-L 之修飾的 gRNA，觀察到類似的結果 (圖4 至 圖 5 以及表 2 及表 3)。此外，觀察到雜質主要為短聚體。The purity of the synthesized modified gRNAs was analyzed. As shown in Figures 2 and 3, similar purity profiles were observed for gRNAs modified according to modification schemes A-F. The purity of these modified gRNAs ranged between 80.1% and 99.5%. Figures 2 and 4 and Table 2 provide purity profiles for gRNAs targeting reference target 1 and modified according to modification schemes A-L, and Figures 3 and 5 and Table 3 provide purity profiles for gRNAs targeting reference target 2 and modified according to modification schemes A-L. As shown in Figures 2 and 3, the sequence of the gRNA had a negligible effect on the purity and ion pair reversed phase liquid chromatography (IPRP) profiles of the modified gRNAs with modifications according to modification schemes A-F. Similar results were observed for gRNAs targeting these two target sequences and with modifications according to modification schemes G-L (Figures 4 to 5 and Tables 2 and 3). In addition, impurities were observed to be mainly short polymers.

如圖4 中所指出，2'-氟修飾顯著增加了 sgRNA 的疏水性。不受特定理論的束縛，較大的疏水性可以能夠實現寡核苷酸之結合以與蛋白質之某些域相互作用且調節該蛋白質之活性 (參見Crooke 等人, Nucleic Acids Research 48(10):5235-5253 (2020)，該文獻之內容藉由引用全文併入本文)。 表 2靶向參考標靶1之gRNA的修飾策略純度(a/a%)理論質量(Da)實驗平均質量(Da)質量差(Da)A98.732428.732426.8-1.9B95.832465.032464.0-1.0C99.532705.132705.0-0.1D98.732312.532312.50.0E80.132571.932572.00.1F98.532327.032326.4-0.6G85.332304.332304.50.2H54.232722.032720.4-1.6I74.032112.732112.0-0.7J75.132300.532300.50.0K74.032342.832342.0-0.8L54.732294.432294.0-0.4表 3靶向參考標靶2之gRNA的修飾策略純度(a/a%)理論質量(Da)實驗平均質量(Da)質量差(Da)A98.832379.732374.3-5.4B98.632416.032415.5-0.5C97.932656.132656.40.3D99.032263.532263.0-0.5E87.932522.832523.00.2F98.232292.032291.5-0.5G79.832255.332255.0-0.3H88.032673.032673.00.0I87.632063.632063.60.0J73.732251.532251.60.1K85.132307.832307.6-0.2L79.132245.432245.50.1As indicated in Figure 4, 2'-fluoro modification significantly increases the hydrophobicity of sgRNA. Without being bound by a particular theory, greater hydrophobicity may enable binding of the oligonucleotide to interact with certain domains of the protein and modulate the activity of the protein (see Crooke et al., Nucleic Acids Research 48(10):5235-5253 (2020), the contents of which are incorporated herein by reference in their entirety). Table 2Modification strategy ofgRNAtargetingreference target1Purity(a/a%)Theoretical quality(Da)Experimental average mass(Da)Quality difference(Da) A 98.7 32428.7 32426.8 -1.9 B 95.8 32465.0 32464.0 -1.0 C 99.5 32705.1 32705.0 -0.1 D 98.7 32312.5 32312.5 0.0 E 80.1 32571.9 32572.0 0.1 F 98.5 32327.0 32326.4 -0.6 G 85.3 32304.3 32304.5 0.2 H 54.2 32722.0 32720.4 -1.6 I 74.0 32112.7 32112.0 -0.7 J 75.1 32300.5 32300.5 0.0 K 74.0 32342.8 32342.0 -0.8 L 54.7 32294.4 32294.0 -0.4 Table 3Modification strategy ofgRNAtargetingreference target2Purity(a/a%)Theoretical quality(Da)Experimental average mass(Da)Quality difference(Da) A 98.8 32379.7 32374.3 -5.4 B 98.6 32416.0 32415.5 -0.5 C 97.9 32656.1 32656.4 0.3 D 99.0 32263.5 32263.0 -0.5 E 87.9 32522.8 32523.0 0.2 F 98.2 32292.0 32291.5 -0.5 G 79.8 32255.3 32255.0 -0.3 H 88.0 32673.0 32673.0 0.0 I 87.6 32063.6 32063.6 0.0 J 73.7 32251.5 32251.6 0.1 K 85.1 32307.8 32307.6 -0.2 L 79.1 32245.4 32245.5 0.1

上述靶向參考標靶 1 且如表 1 中所示進行修飾之 gRNA 分子的功效如下分析。將細胞鋪板於 Prime-XV (IL-7 (25 ng/ml)、IL-15 (50 ng/ml)、TransAct 1:100) 中 (= 第 0 天)。將 4 mg 之各 sgRNA 重懸於 611-624 μL (取決於 MW) 之 IDT Duplex 緩衝液 (目錄編號 1072570) 中，以獲得 200 μM 溶液。使用 3:1 sgRNA:Cas9 比產生 RNP。對於剔除條件，滴定 1.11、3.33、10 及 30 pmol 的 RNP。對於敲入條件，使用 30 pmol 的 RNP，且加入 2 μg 之參考標靶 1-mNeon 奈米質體。按以下次序加入組分：1 – RNP (sgRNA 及 Cas9，首先在室溫下預孵育 15 min)，2 – 模板 (3 μg)，及 3 – 細胞 (2 百萬個)。刺激後 46 小時，在 P3 緩衝液中在 Lonza 4D 電穿孔儀上對細胞進行電穿孔。電穿孔後，將細胞靜置於 37℃ 下 15 min。加入 75 μl 純 Prime-XV，且將細胞轉移至 48 孔盤中不含 TransAct 之 1 ml 完全培養基中。在第 5 天加入 3 ml 完全培養基，然後將細胞轉移至 12 孔盤。在第 5 天取出 0.5 mL 培養物進行 FAC 讀取 (僅 KO 樣品)。在第 6 天取出 0.5 mL 培養物進行 FAC 讀取 (僅 KI 樣品)。隨後在刺激後第 5 天及第 6 天對細胞進行染色，以檢測參考標靶 1 之表現的減少或增加。The efficacy of the gRNA molecules targeting reference target 1 described above and modified as indicated in Table 1 was analyzed as follows. Cells were plated in Prime-XV (IL-7 (25 ng/ml), IL-15 (50 ng/ml), TransAct 1:100) (= day 0). 4 mg of each sgRNA was resuspended in 611-624 μL (depending on MW) of IDT Duplex Buffer (Catalog No. 1072570) to obtain a 200 μM solution. RNPs were generated using a 3:1 sgRNA:Cas9 ratio. For knockout conditions, 1.11, 3.33, 10 and 30 pmol of RNPs were titrated. For knock-in conditions, 30 pmol of RNPs were used, and 2 μg of reference target 1-mNeon nanoplasmids were added. Components were added in the following order: 1 – RNPs (sgRNA and Cas9, first pre-incubated for 15 min at room temperature), 2 – template (3 μg), and 3 – cells (2 million). 46 hours after stimulation, cells were electroporated in P3 buffer on a Lonza 4D electroporator. After electroporation, cells were kept at 37°C for 15 min. 75 μl of pure Prime-XV was added, and cells were transferred to 1 ml of complete medium without TransAct in a 48-well plate. On day 5, 3 ml of complete medium was added and cells were transferred to 12-well plates. On day 5, 0.5 mL of culture was removed for FAC reading (KO samples only). On day 6, 0.5 mL of culture was removed for FAC reading (KI samples only). Cells were then stained on days 5 and 6 after stimulation to detect a decrease or increase in the expression of reference target 1.

圖6 至 圖 12 中所示之經修飾的 gRNA 之編輯效率總結於表 4 及表 5 中。如圖9 及圖 11 以及表 4 中所示，「B」 gRNA 在剔除條件及敲入條件兩者下之表現與參考 gRNA 極相似。gRNA 「D」之表現優於 gRNA 「C」，且 gRNA 「C」之表現優於 gRNA 「A」。在剔除條件及敲入條件兩者下，與參考 gRNA 相比，gRNA 「E」及「F」顯示出極小的活性。The editing efficiencies of the modified gRNAs shown in Figures 6 to 12 are summarized in Tables 4 and 5. As shown in Figures 9 and 11 and Table 4, the performance of "B" gRNA was very similar to the reference gRNA under both knockout and knockin conditions. gRNA "D" performed better than gRNA "C", and gRNA "C" performed better than gRNA "A". gRNAs "E" and "F" showed minimal activity compared to the reference gRNA under both knockout and knockin conditions.

如圖6 至圖 8、圖 10 及圖 12 以及表 5 中所示，與參考 gRNA 相比，gRNA 「G」、「J」及「L」表現出中等程度的剔除活性，但表現出與兩種參考 gRNA 相似的敲入活性。gRNA 「H」及「I」並未導致剔除或敲入活性。此外，與參考 gRNA 相比，gRNA 「K」產生了極少量的剔除或敲入活性。As shown in Figures 6 to 8, 10, and 12 and Table 5, gRNAs "G", "J", and "L" showed moderate knockout activity compared to the reference gRNAs, but showed knockin activity similar to the two reference gRNAs. gRNAs "H" and "I" did not cause knockout or knockin activity. In addition, gRNA "K" produced very little knockout or knockin activity compared to the reference gRNAs.

在經修飾的 gRNA 存在之情況下，測試細胞在剔除條件及敲入條件兩者下的存活率。如圖13 至圖 16 中所示，對於在剔除 (圖13 至圖 14) 條件及敲入 (圖15 至圖 16) 條件下測試之所有經修飾的 gRNA 而言，細胞之存活率極相似。類似地，對於在剔除 (圖13 至圖 14) 條件及敲入 (圖15 至圖 16) 條件下測試之大多數 gRNA 而言，細胞之總數極相似。然而，對於敲入實驗，針對 gRNA 「E」及 gRNA 「F」測得之總細胞更多，此可能係由於此等 gRNA 未能有效切割標靶核酸 (圖15)。 表 4gRNA名稱平均KO活性(10 pmol)平均KI活性(30 pmol)參考 gRNA 289%50%A58%29%B91%47%C80%35%D83%43%E2%0%F4%1%表 5gRNA名稱平均KO活性(10 pmol)平均KI活性(30 pmol)參考 gRNA 277%37%參考 gRNA 370%37%G52%39%H11%0%I6%0%J46%34%K8%1%L38%34%The viability of cells was tested in the presence of modified gRNAs under both knockout and knockin conditions. As shown in Figures 13 to 16, the viability of cells was very similar for all modified gRNAs tested under knockout (Figures 13 to 14) and knockin (Figures 15 to 16) conditions. Similarly, the total number of cells was very similar for most gRNAs tested under knockout (Figures 13 to 14) and knockin (Figures 15 to 16) conditions. However, for the knockin experiments, more total cells were measured for gRNA "E" and gRNA "F", which may be due to the inability of these gRNAs to effectively cleave the target nucleic acid (Figure 15). Table 4gRNANameAverageKOactivity(10 pmol)AverageKIactivity(30 pmol) Reference gRNA 2 89% 50% A 58% 29% B 91% 47% C 80% 35% D 83% 43% E 2% 0% F 4% 1% Table 5gRNANameAverageKOactivity(10 pmol)AverageKIactivity(30 pmol) Reference gRNA 2 77% 37% Reference gRNA 3 70% 37% G 52% 39% H 11% 0% I 6% 0% J 46% 34% K 8% 1% L 38% 34%

如表 4 及表 5 中所總結，與其他所測試的 gRNA 相比，gRNA 「B」、「C」及「D」表現出最大的編輯功效，且與對照組更為相當。實例2：經修飾之gRNA分子的強制降解As summarized in Tables 4 and 5, gRNAs "B", "C", and "D" showed the greatest editing efficacy compared to the other gRNAs tested and were more comparable to the control group.Example2: Forced degradation of modifiedgRNAmolecules

此實例顯示了實例 1 中所述之例示性 gRNA 分子在強制條件下之降解。使實例 1 之 gRNA 分子經受強制降解條件，包括酸性應激 (pH 5)、鹼性應激 (pH 11)、氧化應激及熱應激條件。This example shows the degradation of the exemplary gRNA molecule described in Example 1 under forced conditions. The gRNA molecule of Example 1 was subjected to forced degradation conditions, including acidic stress (pH 5), alkaline stress (pH 11), oxidative stress, and heat stress conditions.

強制降解條件：Forced degradation conditions:

酸性應激(pH 5)：酸性緩衝液：20 mM 乙酸鈉，pH 5.0。溫度：40℃。時間點：第 0 天、第 1 天、第 2 天及第 3 天 (gRNA 「A」至「L」)；第 0 天、第 1 天、第 2 天、第 3 天、第 5 天及第 7 天 (gRNA 「M」至「V」)。Acidic stress(pH 5): Acidic buffer: 20 mM sodium acetate, pH 5.0. Temperature: 40°C. Time points: Day 0, Day 1, Day 2, and Day 3 (gRNA "A" to "L"); Day 0, Day 1, Day 2, Day 3, Day 5, and Day 7 (gRNA "M" to "V").

鹼性應激(pH 11)：鹼性緩衝液：20 mM 碳酸鈉，pH 11.0。溫度：40℃。時間點：第 0 小時、第 8 小時、第 16 小時及第 24 小時 (gRNA 「A」至「L」)；第 0 小時、第 8 小時、第 16 小時、第 24 小時及第 48 小時 (gRNA 「M」至「V」)。Alkaline stress(pH 11): Alkaline buffer: 20 mM sodium carbonate, pH 11.0. Temperature: 40°C. Time points: 0, 8, 16, and 24 hours (gRNA "A" to "L"); 0, 8, 16, 24, and 48 hours (gRNA "M" to "V").

氧化應激：0.3% H₂O₂。 溫度：40℃。時間點：第 0 天、第 1 天、第 2 天、第 3 天及第 5 天 (gRNA 「A」至「V」)。Oxidative stress: 0.3% H₂ O₂ . Temperature: 40°C. Time points: Day 0, Day 1, Day 2, Day 3, and Day 5 (gRNA "A" to "V").

熱應激：Tris-EDTA (TE) 緩衝液，pH 8.0。溫度：40℃。時間點：第 0 天、第 1 天、第 3 天、第 5 天及第 7 天 (gRNA 「A」至「V」)。Heat stress: Tris-EDTA (TE) buffer, pH 8.0. Temperature: 40°C. Time points: Day 0, Day 1, Day 3, Day 5, and Day 7 (gRNA "A" to "V").

樣品製備方法：Sample preparation method:

酸性應激(pH 5)分析：為了製備酸性應激 (pH 5) 溶液，將 164 mg 乙酸鈉加入 100 mL 容量瓶中。將 80 mL LC 級水加入燒瓶中，且用乙酸將 pH 值調整至 5.0。用水使溶液定容。將 10 µL sgRNA 用移液管移入 HPLC 插入管中，且加入 55 µL 酸性應激 (pH 5) 溶液，隨後平緩渦旋 5s。Acid stress(pH 5)analysis: To prepare the acid stress (pH 5) solution, add 164 mg of sodium acetate to a 100 mL volumetric flask. Add 80 mL of LC grade water to the flask and adjust the pH to 5.0 with acetic acid. Bring the solution to volume with water. Pipette 10 µL of sgRNA into an HPLC insert and add 55 µL of the acid stress (pH 5) solution followed by gentle vortexing for 5 s.

鹼性應激(pH 11)分析：為了製備鹼性應激 (pH 11) 溶液，稱取 212 mg 碳酸鈉且將其置於 100 mL 容量瓶中。將約 80 mL LC 級水加入燒瓶中以溶解碳酸鈉，且用 1N HCl 將 pH 值調整至 11.0。用水使溶液定容。將 10 µL sgRNA 用移液管移入 HPLC 插入管中，且加入 55 µL 鹼性應激 (pH 11) 溶液，隨後平緩渦旋 5s。Alkaline stress(pH 11)analysis: To prepare the alkaline stress (pH 11) solution, weigh 212 mg of sodium carbonate and place it in a 100 mL volumetric flask. Add approximately 80 mL of LC-grade water to the flask to dissolve the sodium carbonate and adjust the pH to 11.0 with 1N HCl. Bring the solution to volume with water. Pipette 10 µL of sgRNA into an HPLC insert and add 55 µL of alkaline stress (pH 11) solution followed by gentle vortexing for 5 s.

熱(液體)分析：將 10 µL sgRNA 用移液管移入 HPLC 插入管中，且加入 55 µL TE 緩衝液 (pH 8)，隨後平緩渦旋 5s。Thermal(liquid)analysis: 10 µL of sgRNA was pipetted into an HPLC insert and 55 µL of TE buffer (pH 8) was added, followed by gentle vortexing for 5 s.

氧化分析：為了製備氧化溶液，將 0.59 mL 之 3% H₂O₂加入 5 mL 容量瓶中，且以 TE 緩衝液定容，隨後進行混合。將 10 µL sgRNA 用移液管移入 HPLC 插入管中，且加入 55 µL 之 0.35% H₂O₂，隨後平緩渦旋 5s。Oxidation analysis: To prepare the oxidation solution, add 0.59 mL of 3% H₂ O₂ to a 5 mL volumetric flask and make up to volume with TE buffer, then mix. Pipette 10 µL of sgRNA into an HPLC insert and add 55 µL of 0.35% H₂ O₂ , then vortex gently for 5 s.

HPLC：如表 6 中所示進行 HPLC。 表 6參數條件取樣器溫度40℃管柱DNAPac RP，4 µm，2.1 × 100 mm流速0.40 mL/min管柱溫度70℃移動相A：於水中之 400 mM HFIP + 16.3 mM TEA B：40% MPA+ 60% 甲醇注入體積2 µLUV 波長260 nm梯度-(min) 0 20 21 23 23.5 30MPA：            MPB： 90                    10 40                    60 0                      100 0                      100 90                    10 90                    10HPLC: HPLC was performed as shown in Table 6. Table 6Parameterscondition Sampler temperature 40℃ String DNAPac RP, 4 µm, 2.1 × 100 mm Flow rate 0.40 mL/min Column temperature 70℃ Phase shift A: 400 mM HFIP + 16.3 mM TEA in water B: 40% MPA + 60% methanol Injection volume 2 µL UV wavelength 260 nm Gradient - (min) 0 20 21 23 23.5 30 MPA: MPB: 90 10 40 60 0 100 0 100 90 10 90 10

圖17 顯示了經修飾之 gRNA 在正常條件下的溶析曲線。如圖17 中所示，具有修飾策略 A 至 L 之 gRNA 在時間點 0 (T₀) 時具有範圍在 47%-92% 內的初始純度。此等經修飾之 gRNA 的強制降解研究之結果示於表 7 至表 9 以及圖18 至圖 40 中。 表 7.強制降解下之純度的總結gRNA名稱純度(面積%)T(0)0.3% H₂O₂，40℃，T(5d)pH 8，40℃，T(7d)pH 5，40℃，T(3d)pH 11，40℃，T(1d)參考 gRNA 391.491.286.081.911.2A66.663.762.861.011.8B89.378.382.281.623.0C64.622.461.059.916.6D93.485.088.985.175.9E67.761.0-66.567.0F63.561.360.659.852.6G65.359.459.560.20.8H52.33.947.749.69.9I66.964.463.864.29.3J56.355.252.352.938.2K47.445.842.644.511.7L47.847.745.545.57.0Figure 17 shows the elution curves of the modified gRNA under normal conditions. As shown in Figure 17, the gRNAs with modification strategies A to L have an initial purity ranging from 47% to 92% at time point 0 (T₀ ). The results of the forced degradation studies of these modified gRNAs are shown in Tables 7 to 9 and Figures 18 to 40. Table 7. Summary of Purity under Forced DegradationgRNANamePurity(Area%)T(0)0.3% H₂ O₂,40℃,T(5d)pH 8,40℃,T(7d)pH 5,40℃,T(3d)pH 11,40℃,T(1d) Reference gRNA 3 91.4 91.2 86.0 81.9 11.2 A 66.6 63.7 62.8 61.0 11.8 B 89.3 78.3 82.2 81.6 23.0 C 64.6 22.4 61.0 59.9 16.6 D 93.4 85.0 88.9 85.1 75.9 E 67.7 61.0 - 66.5 67.0 F 63.5 61.3 60.6 59.8 52.6 G 65.3 59.4 59.5 60.2 0.8 H 52.3 3.9 47.7 49.6 9.9 I 66.9 64.4 63.8 64.2 9.3 J 56.3 55.2 52.3 52.9 38.2 K 47.4 45.8 42.6 44.5 11.7 L 47.8 47.7 45.5 45.5 7.0

圖18 顯示了參考 gRNA 3 在鹼性應激 (pH 11)、酸性應激 (pH 5) 及氧化應激 (0.3% H₂O₂) 下之層析圖。圖19 顯示了參考 gRNA 3 在鹼性應激 (pH 11)、酸性應激 (pH 5) 及氧化應激 (0.3% H₂O₂)下歷經某些時間段之層析圖的重疊部分。如圖18 至圖 19 中所示，與導致由於快速降解形成短聚體的鹼性應激 (pH 11) 相比，參考 gRNA 3 對酸水解的抗性更強 (例如，抵抗暴露於酸時之降解)。FIG. 18 shows chromatograms of reference gRNA 3 under alkaline stress (pH 11), acidic stress (pH 5), and oxidative stress (0.3% H₂ O₂ ). FIG. 19 shows an overlay of chromatograms of reference gRNA 3 under alkaline stress (pH 11), acidic stress (pH 5), and oxidative stress (0.3% H₂ O₂ ) over certain time periods. As shown in FIGS. 18-19 , reference gRNA 3 is more resistant to acid hydrolysis (e.g., resists degradation when exposed to acid) than alkaline stress (pH 11), which results in the formation of short polymers due to rapid degradation.

圖20 顯示了 gRNA A、B 及 C 在酸性應激 (pH 5) 下之層析圖。圖21 顯示了經修飾的 gRNA D、E 及 F 在酸性應激 (pH 5) 下之層析圖。圖22 顯示了經修飾的 gRNA G、H 及 I 在酸性應激 (pH 5) 下之層析圖。圖23 顯示了 gRNA J、K 及 L 在酸性應激 (pH 5) 下之層析圖。如圖20 至圖 23 中所示，gRNA E、F、H、I 及 J 對酸性應激的抗性更強。FIG20 shows the chromatograms of gRNA A, B, and C under acidic stress (pH 5). FIG21 shows the chromatograms of modified gRNA D, E, and F under acidic stress (pH 5). FIG22 shows the chromatograms of modified gRNA G, H, and I under acidic stress (pH 5). FIG23 shows the chromatograms of gRNA J, K, and L under acidic stress (pH 5). As shown in FIG20 to FIG23, gRNA E, F, H, I, and J are more resistant to acidic stress.

圖24 顯示了 gRNA A、B 及 C 在鹼性應激 (pH 11) 下之層析圖。圖25 顯示了經修飾的 gRNA D、E 及 F 在鹼性應激 (pH 11) 下之層析圖。圖26 顯示了經修飾的 gRNA G、H 及 I 在鹼性應激 (pH 11) 下之層析圖。圖27 顯示了經修飾的 gRNA J、K 及 L 在鹼性應激 (pH 11) 下之層析圖。如圖24 至圖 27 中所示，經修飾的 gRNA D、E、F、H、I、J 及 K 對鹼性應激 (pH 11) 具有抗性。FIG. 24 shows the chromatograms of gRNA A, B, and C under alkaline stress (pH 11). FIG. 25 shows the chromatograms of modified gRNA D, E, and F under alkaline stress (pH 11). FIG. 26 shows the chromatograms of modified gRNA G, H, and I under alkaline stress (pH 11). FIG. 27 shows the chromatograms of modified gRNA J, K, and L under alkaline stress (pH 11). As shown in FIG. 24 to FIG. 27, modified gRNA D, E, F, H, I, J, and K are resistant to alkaline stress (pH 11).

圖28 顯示了 gRNA A、B 及 C 在熱應激下之層析圖。圖29 顯示了經修飾的 gRNA D、F 及 G 在熱應激下之層析圖。圖30 顯示了經修飾的 gRNA H、I 及 J 在熱應激下之層析圖。圖31 顯示了經修飾的 gRNA K 及 L 在熱應激下之層析圖。如圖28 至圖 31 中所示，大多數 gRNA 對熱降解具有抗性。FIG. 28 shows the chromatograms of gRNA A, B, and C under heat stress. FIG. 29 shows the chromatograms of modified gRNA D, F, and G under heat stress. FIG. 30 shows the chromatograms of modified gRNA H, I, and J under heat stress. FIG. 31 shows the chromatograms of modified gRNA K and L under heat stress. As shown in FIG. 28 to FIG. 31, most gRNAs are resistant to thermal degradation.

圖32 顯示了經修飾的 gRNA A、B 及 C 在氧化應激 (0.3% H₂O₂) 下之層析圖。圖33 顯示了經修飾的 gRNA D、E 及 F 在氧化應激 (0.3% H₂O₂) 下之層析圖。圖34 顯示了經修飾的 gRNA G、H 及 I 在氧化應激 (0.3% H₂O₂) 下之層析圖。圖35 顯示了經修飾的 gRNA J、K 及 L 在氧化應激 (0.3% H₂O₂) 下之層析圖。圖36 顯示了經修飾的 gRNA A 至 L 在氧化應激 (0.3% H₂O₂) 下歷經 5 天時段之層析圖的重疊部分。如圖32 至圖 36 中所示，除了 gRNA C 及 H 之外，gRNA E、G、I、J、K 及 L 對氧化應激的抗性更強。Figure 32 shows the chromatograms of modified gRNA A, B, and C under oxidative stress (0.3% H₂ O₂ ). Figure 33 shows the chromatograms of modified gRNA D, E, and F under oxidative stress (0.3% H₂ O₂ ). Figure 34 shows the chromatograms of modified gRNA G, H, and I under oxidative stress (0.3% H₂ O₂ ). Figure 35 shows the chromatograms of modified gRNA J, K, and L under oxidative stress (0.3% H₂ O₂ ). Figure 36 shows the overlapped portion of the chromatograms of modified gRNA A to L under oxidative stress (0.3% H₂ O₂ ) over a 5-day period. As shown in FIGS. 32 to 36 , in addition to gRNA C and H, gRNA E, G, I, J, K, and L were more resistant to oxidative stress.

圖37 顯示了在酸性應激 (pH 5) 期間歷經 3 天時間段之純度的變化。圖38 顯示了在鹼性應激 (pH 11) 期間歷經 24 小時時段之純度的變化。圖39 顯示了在熱應激期間歷經 7 天時段之純度的變化。如圖39.中所示，沒有足夠的 gRNA E 可用於熱應激研究。圖40 顯示了在氧化應激 (0.3% H₂O₂) 下歷經 5 天時間段之純度的變化。Figure 37 shows the change in purity during acidic stress (pH 5) over a 3-day period. Figure 38 shows the change in purity during alkaline stress (pH 11) over a 24-hour period. Figure 39 shows the change in purity during heat stress over a 7-day period. As shown in Figure 39., there was not enough gRNA E available for the heat stress study. Figure 40 shows the change in purity under oxidative stress (0.3% H₂ O₂ ) over a 5-day period.

如表 7 中所示，許多經修飾的 gRNA 在強制降解條件下抵抗了降解。例如，與參考 gRNA 相比，gRNA 「D」、「E」、「F」及「J」抵抗了降解條件，包括在鹼性應激 (pH 11) 條件下 (表 7)。As shown in Table 7, many of the modified gRNAs resisted degradation under forced degradation conditions. For example, gRNAs "D", "E", "F", and "J" resisted degradation conditions, including under alkaline stress (pH 11), compared to the reference gRNA (Table 7).

表 8 及表 9 提供了對於經修飾的 gRNA 在酸水解 (表 8) 及熱應激 (表 9) 下之純度變化的總結。斜率表明每天純度下降的情況，截距表明純度降低至 0 之天數，且 R 為線性曲線之相關係數。 表 8gRNA名稱線性函數校正係數R參考 gRNA 3y = -3.165x + 91.60.998Ay = -1.692x + 65.5880.967By = -2.708x + 89.2620.986Cy = -1.376x + 64.2140.962Dy = -2.702x + 93.0730.998Ey = -0.125x + 66.9700.965Fy = -1.416x + 63.6840.970Gy = -1.828x + 64.9720.951Hy = -0.65x + 51.5750.990Iy = -0.533x + 66.4370.986Jy = -0.450x + 55.4550.966Ky = -0.754x + 46.8760.964Ly = -1.335x + 49.320.986表 9gRNA名稱線性函數校正係數R參考 gRNA 3y = -0.7998x + 91.7630.990Ay = -0.5379x + 66.7530.991By = -1.0456x + 89.9820.979Cy = -0.4806x + 64.4320.990Dy = -0.6165x + 93.6530.978Fy = -0.4057x + 63.6240.987Gy = -0.6451x + 65.2440.988Hy = -0.6665x + 52.7150.981Iy = -0.4537x + 67.0040.998Jy = -0.5459x + 56.1210.996Ky = -0.6445x + 47.3720.980Ly = -0.2826x + 47.9820.963實例3：gRNA分子之修飾Tables 8 and 9 provide a summary of the purity changes of the modified gRNA under acid hydrolysis (Table 8) and heat stress (Table 9). The slope indicates the purity decreases every day, the intercept indicates the number of days when the purity decreases to 0, and R is the correlation coefficient of the linear curve. Table 8gRNANameLinear functionCorrection coefficientR Reference gRNA 3 y = -3.165x + 91.6 0.998 A y = -1.692x + 65.588 0.967 B y = -2.708x + 89.262 0.986 C y = -1.376x + 64.214 0.962 D y = -2.702x + 93.073 0.998 E y = -0.125x + 66.970 0.965 F y = -1.416x + 63.684 0.970 G y = -1.828x + 64.972 0.951 H y = -0.65x + 51.575 0.990 I y = -0.533x + 66.437 0.986 J y = -0.450x + 55.455 0.966 K y = -0.754x + 46.876 0.964 L y = -1.335x + 49.32 0.986 Table 9gRNANameLinear functionCorrection coefficientR Reference gRNA 3 y = -0.7998x + 91.763 0.990 A y = -0.5379x + 66.753 0.991 B y = -1.0456x + 89.982 0.979 C y = -0.4806x + 64.432 0.990 D y = -0.6165x + 93.653 0.978 F y = -0.4057x + 63.624 0.987 G y = -0.6451x + 65.244 0.988 H y = -0.6665x + 52.715 0.981 I y = -0.4537x + 67.004 0.998 J y = -0.5459x + 56.121 0.996 K y = -0.6445x + 47.372 0.980 L y = -0.2826x + 47.982 0.963Example3:Modification ofgRNA molecules

本實例提供經化學合成以在 gRNA 分子之 5' 端及 3' 端處包括多個修飾的額外 gRNA 分子。如表 10 中所示，本實例提供修飾策略 B 以及 M 至 V。表 10 中所提供之修飾策略 B 為與實例 1 及表 1 所示中之修飾策略 B 相同的策略。表 10 提供了對於此等策略中之各者之修飾的總結，且表 11 提供了對於此等策略中之各者的修飾與參考 gRNA 3 之比較。This example provides additional gRNA molecules that are chemically synthesized to include multiple modifications at the 5' and 3' ends of the gRNA molecules. As shown in Table 10, this example provides modification strategies B and M to V. The modification strategy B provided in Table 10 is the same strategy as the modification strategy B in Example 1 and shown in Table 1. Table 10 provides a summary of the modifications for each of these strategies, and Table 11 provides a comparison of the modifications for each of these strategies with reference gRNA 3.

核苷酸修飾如下在表 10 中指示：*：硫代磷酸酯 (PS) 鍵結；**：二硫代磷酸酯 (PS2) 鍵結；m：2'-OMe；ln：鎖核酸 (LNA)；f：2'-氟；M：2'-O-MOE；以及 d：去氧核糖核苷酸。表 10 之序列中引用的「N」代表任何核苷酸鹼基，例如、鳥嘌呤 (G)、腺嘌呤 (A)、胸腺嘧啶 (T) 或胞嘧啶 (C)。 表 10gRNA名稱5'端處之修飾策略3'端處之修飾策略序列B•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 二硫代磷酸酯鍵結•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  2 個二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個之間)5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3'M•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 二硫代磷酸酯鍵結•  針對末端 3 個核苷酸之 3 個去氧核糖核苷酸 •  2 個二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個之間)5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUdU**dU**dU-3'N•  針對前 3 個連續核苷酸之去氧核糖核苷酸 •  3 二硫代磷酸酯鍵結•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  2 個硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個之間)5'-dN**dN**dN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'O•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 個硫代磷酸酯鍵結•  針對末端 3 個核苷酸之 3 個去氧核糖核苷酸 •  2 個二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個之間)5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUdU**dU**dU-3'P•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 二硫代磷酸酯鍵結•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  2 個硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個之間)5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3'Q•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 個硫代磷酸酯鍵結•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  2 個二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個之間)5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3'R•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  在第一個與第二個核苷酸之間的 1 個硫代磷酸酯鍵結 •  2 個二硫代磷酸酯鍵結 (在第二個與第三個以及第三個與第四個之間)•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  2 個二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個之間)5'-mN*mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3'S•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  2 個二硫代磷酸酯鍵結 (在第一個與第二個之間以及第二個與第三個之間) •  在第三個與第四個核苷酸之間的 1 個硫代磷酸酯鍵結•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  2 個二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間以及在第 n-1 個與第 n-2 個之間)5'-mN**mN**mN*rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3'T•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 二硫代磷酸酯鍵結•  對末端 3 個核苷酸之 2'-O-甲基修飾 •  1 個二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間) •  1 個硫代磷酸酯鍵結 (在第 n-1 個與第 n-2 個之間)5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU**mU-3'U•  對前 4 個連續核苷酸之 2'-O-甲基修飾 •  4 二硫代磷酸酯鍵結•  對末端 5 個核苷酸之 2'-O-甲基修飾 •  4 個二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間、第 n-1 個與第 n-2 個之間、第 n-2 個與第 n-3 個之間以及第 n-3 個與第 n-4 個之間)5'-mN**mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGmC**mU**mU**mU**mU-3'V•  對前 3 個連續核苷酸之 2'-O-甲基修飾 •  3 二硫代磷酸酯鍵結•  對第 n-1 個、第 n-2 個、第 n-3 個及第 n-4 個之 2'-O-甲基修飾。 •  二硫代磷酸酯鍵結 (在第 n 個與第 n-1 個之間、第 n-1 個與第 n-2 個之間以及第 n-2 個與第 n-3 個之間)5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCmU**mU**mU**U-3'表 11gRNA名稱與參考gRNA 3相比，在5'端處的修飾策略與參考gRNA 3相比，在3'端處的修飾策略B•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結M•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結 •  用去氧核糖核苷酸替換經 2'-O-甲基修飾的核苷酸N•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結 •  用去氧核糖核苷酸替換經 2'-O-甲基修飾的核苷酸•  無變化O•  無變化•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結 •  用去氧核糖核苷酸替換經 2'-O-甲基修飾的核苷酸P•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結•  無變化Q•  無變化•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結R•  用二硫代磷酸酯鍵結替換第二個與第三個以及第三個與第四個之間的硫代磷酸酯鍵結•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結S•  用二硫代磷酸酯鍵結替換第一個與第二個之間以及第二個與第三個之間的硫代磷酸酯鍵結•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結T•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結•  用二硫代磷酸酯鍵結替換第 n 個與第 n-1 個之間的硫代磷酸酯鍵結U•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結，且加入額外的二硫代磷酸酯鍵結 (前四個核苷酸具有二硫代磷酸酯鍵結)•  加入兩個額外的 2'-O-甲基修飾的核苷酸 (在第 n-3 個第 n-4 個處) •  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結，且加入兩個額外的二硫代磷酸酯鍵結 (最後四個核苷酸具有二硫代磷酸酯鍵結)V•  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結•  在位置 n 處將經 2'-O-甲基修飾的核苷酸替換為 RNA，且將位置 n-3 處的 RNA 替換為經 2'-O-甲基修飾的核苷酸 •  用二硫代磷酸酯鍵結替換硫代磷酸酯鍵結，且加入額外的二硫代磷酸酯鍵結The nucleotide modifications are indicated in Table 10 as follows: *: phosphorothioate (PS) linkage; **: phosphorodithioate (PS2) linkage; m: 2'-OMe; ln: locked nucleic acid (LNA); f: 2'-fluoro; M: 2'-O-MOE; and d: deoxyribonucleotide. The "N" referenced in the sequences of Table 10 represents any nucleotide base, e.g., guanine (G), adenine (A), thymine (T), or cytosine (C). Table 10gRNAName5'modification strategyModification strategies at the3' endsequence B • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorodithioate bonds • 2'-O-methyl modification of the terminal 3 nucleotides • 2 phosphorodithioate bonds (between nth and n-1th and between n-1th and n-2th) 5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArAr UrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3' M • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorodithioate bonds • 3 deoxyribonucleotides for the terminal 3 nucleotides • 2 phosphorodithioate bonds (between nth and n-1th and between n-1th and n-2th) 5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArAr UrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUdU**dU**dU-3' N • Deoxyribonucleotides for the first 3 consecutive nucleotides • 3 phosphorodithioate bonds • 2'-O-methyl modification of the terminal 3 nucleotides • 2 phosphorothioate bonds (between nth and n-1th and between n-1th and n-2th) 5'-dN**dN**dN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArA rUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3' O • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorothioate bonds • 3 deoxyribonucleotides for the terminal 3 nucleotides • 2 phosphorodithioate bonds (between nth and n-1th and between n-1th and n-2th) 5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArArU rArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUdU**dU**dU-3' P • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorodithioate bonds • 2'-O-methyl modification of the terminal 3 nucleotides • 2 phosphorothioate bonds (between nth and n-1th and between n-1th and n-2th) 5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArA rUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU*mU-3' Q • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorothioate bonds • 2'-O-methyl modification of the terminal 3 nucleotides • 2 phosphorodithioate bonds (between nth and n-1th and between n-1th and n-2th) 5'-mN*mN*mN*rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArArU rArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3' R • 2'-O-methyl modification of the first 3 consecutive nucleotides • 1 phosphorothioate bond between the first and second nucleotides • 2 phosphorodithioate bonds (between the second and third and the third and fourth) • 2'-O-methyl modification of the terminal 3 nucleotides • 2 phosphorodithioate bonds (between nth and n-1th and between n-1th and n-2th) 5'-mN*mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArAr UrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3' S • 2'-O-methyl modification of the first 3 consecutive nucleotides • 2 phosphorodithioate bonds (between the 1st and 2nd and between the 2nd and 3rd) • 1 phosphorothioate bond between the 3rd and 4th nucleotides • 2'-O-methyl modification of the terminal 3 nucleotides • 2 phosphorodithioate bonds (between nth and n-1th and between n-1th and n-2th) 5'-mN**mN**mN*rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArAr UrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU**mU**mU-3' T • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorodithioate bonds • 2'-O-methyl modification of the terminal 3 nucleotides • 1 phosphorodithioate bond (between nth and n-1th) • 1 phosphorothioate bond (between n-1th and n-2th) 5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArA rUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU*mU**mU-3' U • 2'-O-methyl modification of the first 4 consecutive nucleotides • 4 phosphorodithioate bonds • 2'-O-methyl modification of the terminal 5 nucleotides • 4 phosphorodithioate bonds (between n and n-1, n-1 and n-2, n-2 and n-3, and n-3 and n-4) 5'-mN**mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArU rArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGmC**mU**mU**mU**mU-3' V • 2'-O-methyl modification of the first 3 consecutive nucleotides • 3 phosphorodithioate bonds • 2'-O-methyl modification at n-1, n-2, n-3 and n-4. • Phosphorodithioate bonds (between n-1 and n-1, n-1 and n-2 and n-2 and n-3) 5'-mN**mN**mN**rNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrNrGrUrUrUrUrArGrArGrCrUrArGrArArUrArGrCrArArGrUrUrArArArAr UrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCmU**mU**mU**U-3' Table 11gRNAName Modification strategy at the5'endcomparedto referencegRNA 3 Modification strategy at the3'endcomparedto referencegRNA 3 B • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replacement of phosphorothioate bonds with phosphorodithioate bonds M • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replacement of 2'-O-methyl modified nucleotides with deoxyribonucleotides N • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replacement of 2'-O-methyl modified nucleotides with deoxyribonucleotides • No change O • No change • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replacement of 2'-O-methyl modified nucleotides with deoxyribonucleotides P • Replacement of phosphorothioate bonds with phosphorodithioate bonds • No change Q • No change • Replacement of phosphorothioate bonds with phosphorodithioate bonds R • Replacement of phosphorothioate bonds between the second and third and between the third and fourth with phosphorodithioate bonds • Replacement of phosphorothioate bonds with phosphorodithioate bonds S • Replace the phosphorothioate bonds between the first and second and between the second and third with phosphorodithioate bonds • Replacement of phosphorothioate bonds with phosphorodithioate bonds T • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replace the phosphorothioate bond between the nth and n-1th with a phosphorodithioate bond U • Replacement of phosphorothioate bonds with phosphorodithioate bonds, and addition of additional phosphorodithioate bonds (the first four nucleotides have phosphorodithioate bonds) • Addition of two additional 2'-O-methyl modified nucleotides (at positions n-3 and n-4) • Replacement of phosphorothioate bonds with phosphorodithioate bonds and addition of two additional phosphorodithioate bonds (the last four nucleotides have phosphorodithioate bonds) V • Replacement of phosphorothioate bonds with phosphorodithioate bonds • Replacement of a 2'-O-methyl modified nucleotide at position n with RNA, and replacement of RNA at position n-3 with a 2'-O-methyl modified nucleotide • Replacement of phosphorothioate bonds with phosphorodithioate bonds, and addition of additional phosphorodithioate bonds

圖41 及表 12 顯示了參考 gRNA 3 及根據修飾方案 B 及 M 至 V 修飾之 gRNA 的初始純度概況。對於具有根據修飾方案 B 及 M 至 V 之修飾的 gRNA，觀察到類似的純度概況 (表 12 及圖41)。此等經修飾之 gRNA 的強制降解研究之結果示於表 12 及圖41 至圖 59 中。實例 2 中提供了強制降解研究條件的實驗細節。 表 12.強制降解下之純度的總結gRNA名稱純度(面積%)TT(0)00.3% H₂O₂，40℃，T(5d)ppH 8，40℃，T(7d)ppH 5，40℃，T(7d)ppH 11，40℃，T(2d)參考 gRNA 391.286.886.872.618.4B92.583.889.778.840.6M88.176.085.173.549.2N82.779.179.169.544.6O93.478.489.875.759.5P88.977.186.074.373.1Q90.474.187.375.249.8R83.979.180.971.551.6S92.779.789.876.540.9T90.382.786.975.234.7U84.274.281.269.442.4V93.378.589.077.664.6Figure 41 and Table 12 show the initial purity profiles of reference gRNA 3 and gRNAs modified according to modification schemes B and M to V. Similar purity profiles were observed for gRNAs with modifications according to modification schemes B and M to V (Table 12 and Figure 41). The results of the forced degradation studies of these modified gRNAs are shown in Table 12 and Figures 41 to 59. Experimental details of the forced degradation study conditions are provided in Example 2. Table 12. Summary of purity under forced degradationgRNANamePurity(area%)TT(0)00.3% H₂ O₂,40℃,T(5d)ppH 8,40℃,T(7d)ppH 5,40℃,T(7d)ppH 11,40℃,T(2d) Reference gRNA 3 91.2 86.8 86.8 72.6 18.4 B 92.5 83.8 89.7 78.8 40.6 M 88.1 76.0 85.1 73.5 49.2 N 82.7 79.1 79.1 69.5 44.6 O 93.4 78.4 89.8 75.7 59.5 P 88.9 77.1 86.0 74.3 73.1 Q 90.4 74.1 87.3 75.2 49.8 R 83.9 79.1 80.9 71.5 51.6 S 92.7 79.7 89.8 76.5 40.9 T 90.3 82.7 86.9 75.2 34.7 U 84.2 74.2 81.2 69.4 42.4 V 93.3 78.5 89.0 77.6 64.6

圖42 至圖 45 顯示了 gRNA B 及 M 至 V 在酸性應激 (pH 5) 下之層析圖。如圖44 中所示，根據方案「T」修飾之 gRNA 似乎對酸性應激 (pH 5) 條件的抗性最強。此等資料表明，在 gRNA 之 3' 端處包括二硫代磷酸酯鍵結使得在酸性應激條件下之穩定性增加。Figures 42 to 45 show the chromatograms of gRNA B and M to V under acidic stress (pH 5). As shown in Figure 44, the gRNA modified according to protocol "T" appears to be the most resistant to acidic stress (pH 5) conditions. These data suggest that including a phosphorodithioate bond at the 3' end of the gRNA results in increased stability under acidic stress conditions.

圖46 至圖 49 顯示了 gRNA B 及 M 至 V 在鹼性應激 (pH 11) 下之層析圖。如圖47 中所示，根據方案「P」修飾之 gRNA 似乎對鹼性應激 (pH 11) 條件的抗性最強。此等資料表明，在 gRNA 之 5' 端處包括二硫代磷酸酯鍵結使得在鹼性應激條件下之穩定性增加。Figures 46 to 49 show the chromatograms of gRNA B and M to V under alkaline stress (pH 11). As shown in Figure 47, the gRNA modified according to protocol "P" appears to be the most resistant to alkaline stress (pH 11) conditions. These data suggest that including a phosphorodithioate bond at the 5' end of the gRNA results in increased stability under alkaline stress conditions.

圖50 至圖 53 顯示了 gRNA B 及 M 至 V 在熱應激下之層析圖。大多數 gRNA 對熱降解具有抗性。Figures 50 to 53 show the chromatograms of gRNAs B and M to V under heat stress. Most gRNAs are resistant to thermal degradation.

圖54 至圖 55 顯示了 gRNA B 及 M 至 V 在氧化應激 (0.3% H₂O₂) 下之層析圖。大多數 gRNA 易於氧化降解。Figures 54 to 55 show the chromatograms of gRNAs B and M to V under oxidative stress (0.3% H₂ O₂ ). Most gRNAs are susceptible to oxidative degradation.

圖56 顯示了在熱應激期間歷經 7 天時段之純度的變化。圖57 顯示了在酸性應激 (pH 5) 期間歷經 7 天時間段之純度的變化。圖58 顯示了在鹼性應激 (pH 11) 期間歷經 48 小時時段之純度的變化。圖59 顯示了在氧化應激 (0.3% H₂O₂) 下歷經 5 天時間段之純度的變化。Figure 56 shows the change in purity over a 7-day period during heat stress. Figure 57 shows the change in purity over a 7-day period during acidic stress (pH 5). Figure 58 shows the change in purity over a 48-hour period during alkaline stress (pH 11). Figure 59 shows the change in purity over a 5-day period under oxidative stress (0.3% H₂ O₂ ).

在三種供體細胞株 (供體 1、供體 2 及供體 3) 中對靶向參考標靶 1 且根據修飾方案 B 及 M 至 V 修飾之 gRNA 的特徵進行進一步的分析。使用 Lonza 4D 系統，用約 3.0 × 10⁷ 個細胞/tfx 在 100 µL 規模下分析此等 gRNA 之編輯效率、細胞擴增及細胞表型。在供體 1 (圖60 及圖 61)、供體 2 (圖64 及圖 65) 及供體 3 (圖68 及圖 69) 中，在中點及結束點處在根據方案「N」修飾之 gRNA 的情況下，觀察到低編輯率。此外，除了使用方案「N」修飾之 gRNA 之外，對於各經修飾的 gRNA，在所有 3 個供體中均觀察到相似的敲入 (KI) 及剔除 (KO) 編輯百分比。如圖61、圖 65 及圖 69 中所示，與參考 gRNA 相比，在根據修飾方案「S」及「T」修飾之 gRNA 的情況下，觀察到提高的編輯效率。特定而言，在研究結束時間點時，對於參考 gRNA，觀察到野生型細胞 (即未由 gRNA 編輯之細胞) 之數量為 22% 至 39%，而對於「S」及「T」修飾的 gRNA，觀察到野生型細胞之數量在 18% 至 36% 之間。此外，在研究結束時間點時，參考 gRNA 3 之 KO 編輯百分比在 51% 至 78% 之間，與之相比，「S」及「T」修飾的 gRNA 之 KO 編輯百分比在 64% 至 82% 之間，且參考 gRNA 3 之 KI 編輯百分比在 16% 至 23% 之間，與之相比，「S」及「T」修飾的 gRNA 之 KI 編輯百分比在 13% 至 28% 之間。此等結果表明，與參考 gRNA 3 相比，「S」及「T」修飾的 gRNA 表現出相當或提高的編輯效率。gRNAs targeting reference target 1 and modified according to modification protocols B and M to V were further characterized in three donor cell lines (Donor 1, Donor 2, and Donor 3). These gRNAs were analyzed for editing efficiency, cell expansion, and cell phenotype using the Lonza 4D system at a 100 µL scale with approximately 3.0 × 10⁷ cells/tfx. Low editing rates were observed in the case of gRNAs modified according to protocol "N" at the midpoint and end point in Donor 1 (Figures 60 and 61), Donor 2 (Figures 64 and 65), and Donor 3 (Figures 68 and 69). In addition, similar percentages of knock-in (KI) and knock-out (KO) editing were observed in all 3 donors for each modified gRNA, except for the gRNA modified using protocol "N". As shown in Figures 61, 65, and 69, increased editing efficiency was observed in the case of gRNAs modified according to modification protocols "S" and "T" compared to the reference gRNA. Specifically, at the end of the study time point, the number of wild-type cells (i.e., cells not edited by the gRNA) was observed to be 22% to 39% for the reference gRNA, while the number of wild-type cells was observed to be between 18% and 36% for the "S" and "T" modified gRNAs. Furthermore, at the end of the study time points, the KO editing percentages for reference gRNA 3 ranged from 51% to 78%, compared to 64% to 82% for the "S" and "T" modified gRNAs, and the KI editing percentages for reference gRNA 3 ranged from 16% to 23%, compared to 13% to 28% for the "S" and "T" modified gRNAs. These results suggest that the "S" and "T" modified gRNAs exhibit comparable or improved editing efficiencies compared to reference gRNA 3.

對於所有經修飾的 gRNA，在供體 1 (圖62)、供體 2 (圖66) 及供體 3 (圖70) 中，均觀察到相似的細胞擴增速率。 *    *    *    *    *For all modified gRNAs, similar cell proliferation rates were observed in donor 1 (Figure 62), donor 2 (Figure 66), and donor 3 (Figure 70).*    *    *    *    *

本文中提及之所有出版物、專利及專利申請案均藉由引用全文併入本文，其程度就如同每個單獨的出版物、專利或專利申請案被具體地且單獨地指示為藉由引用併入一樣。倘若出現衝突，則以本申請案 (包括本文中之任何定義) 為準。All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

圖1提供本揭露之 gRNA 的例示性結構。圖2提供本揭露之例示性經修飾之 gRNA 的溶析曲線。圖3提供本揭露之例示性經修飾之 gRNA 的溶析曲線。圖4提供本揭露之例示性經修飾之 gRNA 的溶析曲線。圖5提供本揭露之例示性經修飾之 gRNA 的溶析曲線。圖6顯示與參考 gRNA 相比，不同濃度的本揭露之例示性經修飾之 gRNA 的編輯效率。圖7顯示不同濃度的本揭露之例示性經修飾之 gRNA 的編輯效率。圖8顯示與使用來自不同供體之細胞的參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 的編輯效率。圖9顯示與參考 gRNA 相比，不同濃度的本揭露之例示性經修飾之 gRNA 的編輯效率。圖10顯示與參考 gRNA 相比，不同濃度的本揭露之例示性經修飾之 gRNA 的編輯效率。圖11顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 的編輯效率。圖12顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 的編輯效率。圖13提供與參考 gRNA 相比，在剔除條件下在本揭露之例示性經修飾之 gRNA 的存在下之細胞存活百分比及細胞總數。圖14提供與參考 gRNA 相比，在剔除條件下在本揭露之例示性經修飾之 gRNA 的存在下之細胞存活百分比及細胞總數。圖15提供與參考 gRNA 相比，在敲入條件下在本揭露之例示性經修飾之 gRNA 的存在下之細胞存活百分比及細胞總數。圖16提供與參考 gRNA 相比，在剔除條件下在本揭露之例示性經修飾之 gRNA 的存在下之細胞存活百分比及細胞總數。圖17提供本揭露之例示性經修飾之 gRNA 的溶析曲線。圖18提供參考 gRNA 3 在鹼性應激 (pH 11)、酸性應激 (pH 5) 及氧化應激 (0.3% H₂O₂) 條件下之溶析曲線。圖19提供參考 gRNA 3 在鹼性應激 (pH 11)、酸性應激 (pH 5) 及氧化應激 (0.3% H₂O₂) 條件下歷經某些時間段之溶析曲線。圖20提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的溶析曲線。圖21提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的溶析曲線。圖22提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的溶析曲線。圖23提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的溶析曲線。圖24提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的溶析曲線。圖25提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的溶析曲線。圖26提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的溶析曲線。圖27提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的溶析曲線。圖28提供本揭露之例示性經修飾之 gRNA在熱應激下的溶析曲線。圖29提供本揭露之例示性經修飾之 gRNA在熱應激下的溶析曲線。圖30提供本揭露之例示性經修飾之 gRNA在熱應激下的溶析曲線。圖31提供本揭露之例示性經修飾之 gRNA在熱應激下的溶析曲線。圖32提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下的溶析曲線。圖33提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下的溶析曲線。圖34提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下的溶析曲線。圖35提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下的溶析曲線。圖36提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下歷經 5 天時段的溶析曲線。圖37提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下歷經 3 天時間段的純度變化。圖38提供本揭露之例示性經修飾之 gRNA 在鹼性應激 (pH 11) 下歷經 24 小時時間段的純度變化。圖39提供本揭露之例示性經修飾之 gRNA 在熱應激下歷經 7 天時間段的純度變化。圖40提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下歷經 5 天時間段的純度變化。圖41提供本揭露之例示性經修飾之 gRNA 的溶析曲線。圖42提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的溶析曲線。圖43提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的溶析曲線。圖44提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的溶析曲線。圖45提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的溶析曲線。圖46提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的溶析曲線。圖47提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的溶析曲線。圖48提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的溶析曲線。圖49提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的溶析曲線。圖50提供本揭露之例示性經修飾之 gRNA在熱應激下的溶析曲線。圖51提供本揭露之例示性經修飾之 gRNA在熱應激下的溶析曲線。圖52提供本揭露之例示性經修飾之 gRNA在熱應激下的溶析曲線。圖53提供本揭露之例示性經修飾之 gRNA在熱應激下的溶析曲線。圖54提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下的溶析曲線。圖55提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下的溶析曲線。圖56提供本揭露之例示性經修飾之 gRNA 在熱應激下歷經 7 天時間段的純度變化。圖57提供本揭露之例示性經修飾之 gRNA 在酸性應激 (pH 5) 條件下的純度變化。圖58提供本揭露之例示性經修飾之 gRNA在鹼性應激 (pH 11) 條件下的純度變化。圖59提供本揭露之例示性經修飾之 gRNA 在氧化應激 (0.3% H₂O₂) 下的純度變化。圖60顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 在中間時間點時的編輯效率。圖61顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 在結束時間點時的編輯效率。圖62顯示供體 1 之細胞在用本揭露之例示性經修飾之 gRNA 轉染 (Tfx) 後在第 5 天及自第 5 天至第 12 天的細胞擴增。圖63顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 在結束時間點時的表型。圖64顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 在中間時間點時的編輯效率。圖65顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 在結束時間點時的編輯效率。圖66顯示供體 2 之細胞在用本揭露之例示性經修飾之 gRNA 轉染 (Tfx) 後在第 5 天及自第 5 天至第 12 天的細胞擴增。圖67顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 在結束時間點時的表型。圖68顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 在中間時間點時的編輯效率。圖69顯示與參考 gRNA 相比，本揭露之例示性經修飾之 gRNA 在結束時間點時的編輯效率。圖70顯示供體 3 之細胞在用本揭露之例示性經修飾之 gRNA 轉染 (Tfx) 後在第 5 天及自第 5 天至第 12 天的細胞擴增。Figure1 provides an exemplary structure of a gRNA disclosed herein.Figure2 provides an elution curve of an exemplary modified gRNA disclosed herein.Figure3 provides an elution curve of an exemplary modified gRNA disclosed herein.Figure4 provides an elution curve of an exemplary modified gRNA disclosed herein.Figure5 provides an elution curve of an exemplary modified gRNA disclosed herein.Figure6 shows the editing efficiency of an exemplary modified gRNA disclosed herein at different concentrations compared to a reference gRNA.Figure7 shows the editing efficiency of an exemplary modified gRNA disclosed herein at different concentrations.Figure8 shows the editing efficiency of an exemplary modified gRNA disclosed herein compared to a reference gRNA using cells from different donors.Figure9 shows the editing efficiency of exemplary modified gRNAs of the present disclosure at different concentrations compared to a reference gRNA.Figure10 shows the editing efficiency of exemplary modified gRNAs of the present disclosure at different concentrations compared to a reference gRNA.Figure11 shows the editing efficiency of exemplary modified gRNAs of the present disclosure compared to a reference gRNA.Figure12 shows the editing efficiency of exemplary modified gRNAs of the present disclosure compared to a reference gRNA.Figure13 provides the percentage of cell survival and the total number of cells in the presence of exemplary modified gRNAs of the present disclosure under knockout conditions compared to a reference gRNA.Figure14 provides the percentage of cell survival and the total number of cells in the presence of exemplary modified gRNAs of the present disclosure under knockout conditions compared to a reference gRNA.FIG.15 provides the percentage of cell survival and the total number of cells in the presence of an exemplary modified gRNA of the present disclosure under knock-in conditions compared to a reference gRNA.FIG .16 provides the percentage of cell survival and the total number of cells in the presence of an exemplary modified gRNA of the present disclosure under knock-out conditions compared to a reference gRNA.FIG.17 provides the elution curves of an exemplary modified gRNA of the present disclosure.FIG.18 provides the elution curves of reference gRNA 3 under alkaline stress (pH 11), acidic stress (pH 5), and oxidative stress (0.3% H₂ O₂ ) conditions.FIG.19 provides the elution curves of reference gRNA 3 under alkaline stress (pH 11), acidic stress (pH 5), and oxidative stress (0.3% H₂ O₂ ) conditions over certain time periods.FIG.20 provides the elution curves of exemplary modified gRNAs of the present disclosure under acidic stress (pH 5).FIG.21 provides the elution curves of exemplary modified gRNAs of the present disclosure under acidic stress (pH 5).FIG.22 provides the elution curves of exemplary modified gRNAs of the present disclosure under acidic stress (pH 5).FIG.23 provides the elution curves of exemplary modified gRNAs of the present disclosure under acidic stress (pH 5).Figure24 provides an exemplary modified gRNA dissolution curve under alkaline stress (pH 11) conditions.Figure25 provides an exemplary modified gRNA dissolution curve under alkaline stress (pH 11) conditions.Figure26 provides an exemplary modified gRNA dissolution curve under alkaline stress (pH 11) conditions.Figure27 provides an exemplary modified gRNA dissolution curve under alkaline stress (pH 11) conditions.Figure28 provides an exemplary modified gRNA dissolution curve under heat stress.Figure29 provides an exemplary modified gRNA dissolution curve under heat stress.Figure30 provides an exemplary modified gRNA dissolution curve under heat stress.Figure31 provides the dissolution curve of the exemplary modified gRNA of the present disclosure under heat stress.Figure32 provides the dissolution curve of the exemplary modified gRNA of the present disclosure under oxidative stress (0.3% H₂ O₂ ).Figure33 provides the dissolution curve of the exemplary modified gRNA of the present disclosure under oxidative stress (0.3% H₂ O₂ ).Figure34 provides the dissolution curve of the exemplary modified gRNA of the present disclosure under oxidative stress (0.3% H₂ O₂ ).Figure35 provides the dissolution curve of the exemplary modified gRNA of the present disclosure under oxidative stress (0.3% H₂ O₂ ).Figure36 provides the dissolution curve of the exemplary modified gRNA of the present disclosure under oxidative stress (0.3% H₂ O₂ ) over a period of 5 days.Figure37 provides the purity change of exemplary modified gRNA of the present disclosure under acidic stress (pH 5) over a 3-day period.Figure38 provides the purity change of exemplary modified gRNA of the present disclosure under alkaline stress (pH 11) over a 24-hour period.Figure39 provides the purity change of exemplary modified gRNA of the present disclosure under heat stress over a 7-day period.Figure40 provides the purity change of exemplary modified gRNA of the present disclosure under oxidative stress (0.3% H₂ O₂ ) over a 5-day period.Figure41 provides the elution curve of exemplary modified gRNA of the present disclosure.Figure42 provides the elution curve of exemplary modified gRNA of the present disclosure under acidic stress (pH 5).Figure43 provides an elution curve of an exemplary modified gRNA of the present disclosure under acidic stress (pH 5).Figure44 provides an elution curve of an exemplary modified gRNA of the present disclosure under acidic stress (pH 5).Figure45 provides an elution curve of an exemplary modified gRNA of the present disclosure under acidic stress (pH 5).Figure46 provides an elution curve of an exemplary modified gRNA of the present disclosure under alkaline stress (pH 11).Figure47 provides an elution curve of an exemplary modified gRNA of the present disclosure under alkaline stress (pH 11).Figure48 provides an elution curve of an exemplary modified gRNA of the present disclosure under alkaline stress (pH 11).Figure49 provides an exemplary modified gRNA dissolution curve under alkaline stress (pH 11) conditions.Figure50 provides an exemplary modified gRNA dissolution curve under heat stress.Figure51 provides an exemplary modified gRNA dissolution curve under heat stress.Figure52 provides an exemplary modified gRNA dissolution curve under heat stress.Figure53 provides an exemplary modified gRNA dissolution curve under heat stress.Figure54 provides an exemplary modified gRNA dissolution curve under oxidative stress (0.3% H₂ O₂ ).Figure55 provides an exemplary modified gRNA dissolution curve under oxidative stress (0.3% H₂ O₂ ).Figure56 provides the purity change of exemplary modified gRNA of the present disclosure under heat stress over a 7-day period.Figure57 provides the purity change of exemplary modified gRNA of the present disclosure under acidic stress (pH 5).Figure58 provides the purity change of exemplary modified gRNA of the present disclosure under alkaline stress (pH 11).Figure59 provides the purity change of exemplary modified gRNA of the present disclosure under oxidative stress (0.3% H₂ O₂ ).Figure60 shows the editing efficiency of exemplary modified gRNA of the present disclosure at the intermediate time point compared to the reference gRNA.Figure61 shows the editing efficiency of exemplary modified gRNA of the present disclosure at the end time point compared to the reference gRNA.Figure62 shows cell expansion of donor 1 on day 5 and from day 5 to day 12 after transfection (Tfx) with an exemplary modified gRNA of the present disclosure.Figure63 shows the phenotype of the exemplary modified gRNA of the present disclosure at the end time point compared to the reference gRNA.Figure64 shows the editing efficiency of the exemplary modified gRNA of the present disclosure at the intermediate time point compared to the reference gRNA.Figure65 shows the editing efficiency of the exemplary modified gRNA of the present disclosure at the end time point compared to the reference gRNA.Figure66 shows cell expansion of donor 2 on day 5 and from day 5 to day 12 after transfection (Tfx) with an exemplary modified gRNA of the present disclosure.Figure67 shows the phenotype of exemplary modified gRNAs of the present disclosure at the end time point compared to the reference gRNA.Figure68 shows the editing efficiency of exemplary modified gRNAs of the present disclosure at the intermediate time point compared to the reference gRNA.Figure69 shows the editing efficiency of exemplary modified gRNAs of the present disclosure at the end time point compared to the reference gRNA.Figure70 shows cell expansion of donor 3 cells at day 5 and from day 5 to day 12 after transfection (Tfx) with exemplary modified gRNAs of the present disclosure.

Claims (53)

Translated fromChinese

一種引導 RNA 分子，其包含： (a) 在該引導 RNA 分子的 5’ 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結； (b) 在該引導 RNA 分子的 3’ 端 (第「n」個) 核苷酸與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結；或 (c) 在該引導 RNA 分子的 5’ 端處的第一個與第二個核苷酸之間的二硫代磷酸酯鍵結以及在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸之間的二硫代磷酸酯鍵結。A guide RNA molecule comprising:(a) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule;(b) a phosphorodithioate bond between the 3' ("nth") nucleotide and the n-1th nucleotide of the guide RNA molecule; or(c) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule and a phosphorodithioate bond between the nth and n-1th nucleotides of the guide RNA molecule.如請求項 1 之引導 RNA 分子，其進一步包含在該引導 RNA 分子的 5’ 端處的第二個與第三個核苷酸以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。The guide RNA molecule of claim 1, further comprising dithioate bonds between the second and third nucleotides and the third and fourth nucleotides at the 5' end of the guide RNA molecule.如請求項 1 或 2 之引導 RNA 分子，其包含在該引導 RNA 分子的 5’ 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、以及第三個與第四個核苷酸之間的二硫代磷酸酯鍵結。A guide RNA molecule as claimed in claim 1 or 2, comprising dithiophosphate bonds between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the guide RNA molecule.如請求項 1 至 3 中任一項之引導 RNA 分子，其包含在該引導 RNA 分子的 5’ 端處的第一個與第二個核苷酸、第二個與第三個核苷酸、第三個與第四個核苷酸、以及第四個與第五個核苷酸之間的二硫代磷酸酯鍵結。A guide RNA molecule as in any one of claims 1 to 3, comprising dithiophosphate bonds between the first and second nucleotides, the second and third nucleotides, the third and fourth nucleotides, and the fourth and fifth nucleotides at the 5' end of the guide RNA molecule.如請求項 1 至 4 中任一項之引導 RNA 分子，其包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸以及第 n-1 個與第 n-2 個核苷酸或者第 n 個與第 n-1 個核苷酸以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。A guide RNA molecule as in any one of claims 1 to 4, comprising a phosphorothioate bond between the nth and n-1th nucleotides and the n-1th and n-2th nucleotides or between the nth and n-1th nucleotides and the n-2th and n-3th nucleotides of the guide RNA molecule.如請求項 1 至 5 中任一項之引導 RNA 分子，其包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸、第 n-1 個與第 n-2 個核苷酸、以及第 n-2 個與第 n-3 個核苷酸之間的二硫代磷酸酯鍵結。A guide RNA molecule as in any one of claims 1 to 5, comprising dithiophosphate bonds between the nth and n-1th nucleotides, the n-1th and n-2th nucleotides, and the n-2th and n-3th nucleotides of the guide RNA molecule.如請求項 1 至 6 中任一項之引導 RNA 分子，其包含在該引導 RNA 分子的第 n 個與第 n-1 個核苷酸、第 n-1 個與第 n-2 個核苷酸、第 n-2 個與第 n-3 個核苷酸、以及第 n-3 個與第 n-4 個核苷酸之間的二硫代磷酸酯鍵結。A guide RNA molecule as in any one of claims 1 to 6, comprising dithiophosphate bonds between the n-th and n-1-th nucleotides, the n-1-th and n-2-th nucleotides, the n-2-th and n-3-th nucleotides, and the n-3-th and n-4-th nucleotides of the guide RNA molecule.如請求項 1 至 7 中任一項之引導 RNA 分子，其中在該引導 RNA 分子的 5’ 端處的第一個核苷酸包含選自由以下所組成之群組的修飾： (a) 經 2’-氟修飾的核苷酸； (b) 經 2’-O-甲基修飾的核苷酸； (c) 經 2'-O-(2-甲氧基乙基) 修飾的核苷酸； (d) 鎖核酸 (LNA)； (e) 去氧核糖核苷酸；以及 (f) (a) 至 (e) 中之兩者或更多者之組合。A guide RNA molecule as claimed in any one of claims 1 to 7, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a modification selected from the group consisting of:(a) a 2'-fluorine-modified nucleotide;(b) a 2'-O-methyl-modified nucleotide;(c) a 2'-O-(2-methoxyethyl)-modified nucleotide;(d) a locked nucleic acid (LNA);(e) a deoxyribonucleotide; and(f) a combination of two or more of (a) to (e).如請求項 8 之引導 RNA 分子，其中在該引導 RNA 分子的 5’ 端處的第一個核苷酸包含經 2'-氟修飾的核苷酸。The guide RNA molecule of claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-fluorine-modified nucleotide.如請求項 8 之引導 RNA 分子，其中在該引導 RNA 分子的 5’ 端處的第一個核苷酸包含經 2'-O-甲基修飾的核苷酸。The guide RNA molecule of claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-O-methyl modified nucleotide.如請求項 8 之引導 RNA 分子，其中在該引導 RNA 分子的 5’ 端處的第一個核苷酸包含經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。The guide RNA molecule of claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl)-modified nucleotide.如請求項 8 之引導 RNA 分子，其中在該引導 RNA 分子的 5’ 端處的第一個核苷酸包含 LNA。The guide RNA molecule of claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises LNA.如請求項 8 之引導 RNA 分子，其中在該引導 RNA 分子的 5’ 端處的第一個核苷酸包含去氧核糖核苷酸。The guide RNA molecule of claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a deoxyribonucleotide.如請求項 1 至 13 中任一項之引導 RNA 分子，其中在該引導 RNA 分子的 5’ 端處的 (i) 第二個核苷酸、(ii) 第三個核苷酸、(iii) 第四個核苷酸、(iv) 第二個與第三個核苷酸、(v) 第二個與第四個核苷酸、(vi) 第三個與第四個核苷酸或 (v) 第二個、第三個與第四個核苷酸包含選自由以下所組成之群組的修飾： (a) 經 2’-氟修飾的核苷酸； (b) 經 2’-O-甲基修飾的核苷酸； (c) 經 2’-O-(2-甲氧基乙基) 修飾的核苷酸； (d) 鎖核酸 (LNA)； (e) 去氧核糖核苷酸；以及 (f) (a) 至 (e) 中之兩者或更多者之組合。A guide RNA molecule as claimed in any one of claims 1 to 13, wherein (i) the second nucleotide, (ii) the third nucleotide, (iii) the fourth nucleotide, (iv) the second and third nucleotides, (v) the second and fourth nucleotides, (vi) the third and fourth nucleotides, or (v) the second, third and fourth nucleotides at the 5' end of the guide RNA molecule comprise a modification selected from the group consisting of:(a) 2'-fluorine-modified nucleotides;(b) 2'-O-methyl-modified nucleotides;(c) 2'-O-(2-methoxyethyl)-modified nucleotides;(d) locked nucleic acids (LNA);(e) deoxyribonucleotides; and(f) a combination of two or more of (a) to (e).如請求項 1 至 9 及 14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5’ 端處的前三個核苷酸處包含三個連續的經 2’-氟修飾的核苷酸。A guide RNA molecule as in any one of claims 1 to 9 and 14, wherein the guide RNA molecule comprises three consecutive 2'-fluorine-modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.如請求項 1 至 8、10 及 14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5’ 端處的前三個核苷酸處包含三個連續的經 2’-O-甲基修飾的核苷酸。A guide RNA molecule as in any one of claims 1 to 8, 10 and 14, wherein the guide RNA molecule comprises three consecutive 2'-O-methyl modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.如請求項 1 至 8、10 及 14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5’ 端處的前四個核苷酸處包含四個連續的經 2’-O-甲基修飾的核苷酸。A guide RNA molecule as in any one of claims 1 to 8, 10 and 14, wherein the guide RNA molecule comprises four consecutive 2'-O-methyl modified nucleotides at the first four nucleotides at the 5' end of the guide RNA molecule.如請求項 1 至 8、11 及 14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5’ 端處的前三個核苷酸處包含三個連續的經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。A guide RNA molecule as claimed in any one of claims 1 to 8, 11 and 14, wherein the guide RNA molecule comprises three consecutive 2'-O-(2-methoxyethyl)-modified nucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.如請求項 1 至 8、12 及 14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5’ 端處的前三個核苷酸處包含三個連續的 LNA。A guide RNA molecule as in any one of claims 1 to 8, 12 and 14, wherein the guide RNA molecule comprises three consecutive LNAs at the first three nucleotides at the 5' end of the guide RNA molecule.如請求項 1 至 8、13 及 14 中任一項之引導 RNA 分子，其中該引導 RNA 分子在該引導 RNA 分子的 5’ 端處的前三個核苷酸處包含三個連續的去氧核糖核苷酸。A guide RNA molecule as in any one of claims 1 to 8, 13 and 14, wherein the guide RNA molecule comprises three consecutive deoxyribonucleotides at the first three nucleotides at the 5' end of the guide RNA molecule.如請求項 1 至 20 中任一項之引導 RNA 分子，其中該引導 RNA 分子的 3’ 端 (第「n」個) 核苷酸包含選自由以下所組成之群組的修飾： (a) 經 2’-氟修飾的核苷酸； (b) 經 2’-O-甲基修飾的核苷酸； (c) 經 2’-O-(2-甲氧基乙基) 修飾的核苷酸； (d) 鎖核酸 (LNA)； (e) 去氧核糖核苷酸；以及 (f) (a) 至 (e) 中之兩者或更多者之組合。A guide RNA molecule as claimed in any one of claims 1 to 20, wherein the 3' end ("n"th) nucleotide of the guide RNA molecule comprises a modification selected from the group consisting of:(a) 2'-fluorine modified nucleotides;(b) 2'-O-methyl modified nucleotides;(c) 2'-O-(2-methoxyethyl) modified nucleotides;(d) locked nucleic acids (LNA);(e) deoxyribonucleotides; and(f) a combination of two or more of (a) to (e).如請求項 21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含經 2'-氟修飾的核苷酸。A guide RNA molecule as claimed in claim 21, wherein the nth nucleotide of the guide RNA molecule comprises a 2'-fluorine-modified nucleotide.如請求項 21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含經 2’-O-甲基修飾的核苷酸。A guide RNA molecule as claimed in claim 21, wherein the nth nucleotide of the guide RNA molecule comprises a 2’-O-methyl modified nucleotide.如請求項 21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含經 2’-O-(2-甲氧基乙基) 修飾的核苷酸。The guide RNA molecule of claim 21, wherein the nth nucleotide of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl)-modified nucleotide.如請求項 21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含 LNA。The guide RNA molecule of claim 21, wherein the nth nucleotide of the guide RNA molecule comprises LNA.如請求項 21 之引導 RNA 分子，其中該引導 RNA 分子的第 n 個核苷酸包含去氧核糖核苷酸。A guide RNA molecule as claimed in claim 21, wherein the nth nucleotide of the guide RNA molecule comprises a deoxyribonucleotide.如請求項 1 至 26 中任一項之引導 RNA 分子，其中該引導 RNA 分子的 (i) 第 n-1 個核苷酸、(ii) 第 n-2 個核苷酸、(iii) 第 n-3 個核苷酸、(iv) 第 n-4 個核苷酸、(v) 第 n 個與第 n-1 個核苷酸、(vi) 第 n 個與第 n-2 個核苷酸、(vii) 第 n-1 個與第 n-2 個核苷酸、(viii) 第 n 個、第 n-1 個與第 n-2 個核苷酸、(ix) 第 n-1 個、第 n-2 個與第 n-3 個核苷酸、(x) 第 n 個、第 n-1 個、第 n-2 個與第 n-3 個核苷酸、或 (xi) 第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自包含選自由以下所組成之群組的修飾： (a) 經 2’-氟修飾的核苷酸； (b) 經 2’-O-甲基修飾的核苷酸； (c) 經 2’-O-(2-甲氧基乙基) 修飾的核苷酸； (d) 鎖核酸 (LNA)； (e) 去氧核糖核苷酸；以及 (f) (a) 至 (e) 中之兩者或更多者之組合。The guide RNA molecule of any one of claims 1 to 26, wherein (i) the n-1th nucleotide, (ii) the n-2th nucleotide, (iii) the n-3th nucleotide, (iv) the n-4th nucleotide, (v) the n-th and n-1th nucleotides, (vi) the n-th and n-2th nucleotides, (vii) the n-1th and n-2th nucleotides, (viii) the n-th, n-1th and n-2th nucleotides, (ix) the n-1th, n-2th and n-3th nucleotides, (x) the n-th, n-1th, n-2th and n-3th nucleotides, or (xi) the n-th, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule. Each of the nucleotides comprises a modification selected from the group consisting of:(a) 2’-fluorine-modified nucleotides;(b) 2’-O-methyl-modified nucleotides;(c) 2’-O-(2-methoxyethyl)-modified nucleotides;(d) locked nucleic acids (LNA);(e) deoxyribonucleotides; and(f) a combination of two or more of (a) to (e).如請求項 1 至 22 及 27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-氟修飾的核苷酸。The guide RNA molecule of any one of claims 1 to 22 and 27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-fluorine-modified nucleotides.如請求項 1 至 21、23 及 27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-O-甲基修飾的核苷酸。The guide RNA molecule of any one of claims 1 to 21, 23 and 27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-O-methyl modified nucleotides.如請求項 1 至 21、23、27 及 29 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-O-甲基修飾的核苷酸。A guide RNA molecule as claimed in any one of claims 1 to 21, 23, 27 and 29, wherein the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-O-methyl modified nucleotides.如請求項 1 至 21、24 及 27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為經 2'-O-(2-甲氧基乙基) 修飾的核苷酸。The guide RNA molecule of any one of claims 1 to 21, 24 and 27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each 2'-O-(2-methoxyethyl)-modified nucleotides.如請求項 1 至 21、25 及 27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為 LNA。The guide RNA molecule of any one of claims 1 to 21, 25 and 27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each LNA.如請求項 1 至 21、26 及 27 中任一項之引導 RNA 分子，其中該引導 RNA 分子的第 n 個與第 n-1 個核苷酸，第 n 個與第 n-2 個核苷酸，第 n-1 個與第 n-2 個核苷酸，第 n 個、第 n-1 個與第 n-2 個核苷酸，第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸，或第 n 個、第 n-1 個、第 n-2 個、第 n-3 個與第 n-4 個核苷酸各自為去氧核糖核苷酸。The guide RNA molecule of any one of claims 1 to 21, 26 and 27, wherein the nth and n-1th nucleotides, the nth and n-2th nucleotides, the n-1th and n-2th nucleotides, the nth, n-1th and n-2th nucleotides, the n-1th, n-2th, n-3th and n-4th nucleotides, or the nth, n-1th, n-2th, n-3th and n-4th nucleotides of the guide RNA molecule are each deoxyribonucleotides.一種核酸，其包含編碼如請求項 1 至 33 中任一項之引導 RNA 分子的多核苷酸。A nucleic acid comprising a polynucleotide encoding a guide RNA molecule as described in any one of claims 1 to 33.如請求項 34 之核酸，其進一步包含編碼 RNA 引導的核酸酶的多核苷酸。The nucleic acid of claim 34, further comprising a polynucleotide encoding an RNA-guided nuclease.如請求項 35 之核酸，其中該 RNA 引導的核酸酶為 Cas 蛋白。The nucleic acid of claim 35, wherein the RNA-guided nuclease is a Cas protein.如請求項 36 之核酸，其中該 Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。The nucleic acid of claim 36, wherein the Cas protein is selected from the group consisting of: Cas9, Cas12, Cas13 and combinations thereof.一種載體，其包含如請求項 34 至 37 中任一項之核酸。A vector comprising the nucleic acid of any one of claims 34 to 37.一種組成物，其包含如請求項 1 至 33 中任一項之引導 RNA 分子。A composition comprising a guide RNA molecule as claimed in any one of claims 1 to 33.如請求項 39 之組成物，其進一步包含 RNA 引導的核酸酶。The composition of claim 39, further comprising an RNA-guided nuclease.如請求項 39 之組成物，其進一步包含編碼該 RNA 引導的核酸酶的核酸。The composition of claim 39, further comprising a nucleic acid encoding the RNA-guided nuclease.如請求項 40 或 41 之組成物，其中該 RNA 引導的核酸酶為 Cas 蛋白。The composition of claim 40 or 41, wherein the RNA-guided nuclease is a Cas protein.一種組成物，其包含如請求項 34 至 37 中任一項之核酸。A composition comprising the nucleic acid of any one of claims 34 to 37.一種組成物，其包含如請求項 38 之載體。A composition comprising the carrier of claim 38.一種核糖核酸蛋白 (RNP) 複合物，其包含如請求項 1 至 33 中任一項之引導 RNA 分子以及 RNA 引導的核酸酶。A ribonucleoprotein (RNP) complex comprising a guide RNA molecule as claimed in any one of claims 1 to 33 and an RNA-guided nuclease.如請求項 45 之 RNP 複合物，其中該 RNA 引導的核酸酶為 Cas 蛋白。The RNP complex of claim 45, wherein the RNA-guided nuclease is a Cas protein.如請求項 46 之 RNP 複合物，其中該 Cas 蛋白係選自由以下所組成之群組：Cas9、Cas12、Cas13 及其組合。The RNP complex of claim 46, wherein the Cas protein is selected from the group consisting of: Cas9, Cas12, Cas13 and a combination thereof.一種細胞，其包含如請求項 39 至 44 中任一項之組成物。A cell comprising the composition of any one of claims 39 to 44.一種細胞，其包含如請求項 45 至 47 中任一項之 RNP 複合物。A cell comprising the RNP complex of any one of claims 45 to 47.一種修飾細胞之方法，其包含使該細胞與如請求項 39 至 44 中任一項之組成物或如請求項 45 至 47 中任一項之 RNP 複合物接觸。A method for modifying a cell, comprising contacting the cell with a composition as described in any one of claims 39 to 44 or an RNP complex as described in any one of claims 45 to 47.如請求項 50 之方法，其中該接觸包含藉由電穿孔將該組成物引入該細胞中。The method of claim 50, wherein the contacting comprises introducing the composition into the cell by electroporation.一種治療有需要之個體之方法，其包含： (a) 藉由使該個體的細胞與如請求項 39 至 44 中任一項之組成物或如請求項 45 至 47 中任一項之 RNP 複合物接觸，離體修飾該細胞；以及 (b) 將經修飾的細胞送回至該個體。A method for treating an individual in need thereof, comprising:(a) modifying a cell of the individual ex vivo by contacting the cell with a composition as in any one of claims 39 to 44 or an RNP complex as in any one of claims 45 to 47; and(b) returning the modified cell to the individual.一種基因編輯系統，其包含： (a) 一或多個如請求項 1 至 33 中任一項之引導 RNA 分子； (b) 一或多個如請求項 34 至 37 中任一項之核酸； (c) 一或多個如請求項 38 之載體； (d) 一或多個如請求項 39 至 44 中任一項之組成物；及/或 (e) 一或多個如請求項 45 至 47 中任一項之 RNP 複合物。A gene editing system comprising:(a) one or more guide RNA molecules as described in any one of claims 1 to 33;(b) one or more nucleic acids as described in any one of claims 34 to 37;(c) one or more vectors as described in claim 38;(d) one or more compositions as described in any one of claims 39 to 44; and/or(e) one or more RNP complexes as described in any one of claims 45 to 47.