[0065] As an alternative to the holo Cascade complex, subcomplexes of Cascade that are missing either CasA (-A) or CasA. and CasB (-A-B) were purified (Figure 2A). These smaller subcomplexes are sensible alternatives that may be more amenable to high-resolution structure determination. Each of the individual subunits may also be purified . As an alternative to the Cascade complex from E. coli, we have also developed a method for purifying the Cascade complex from T. thermophilus (Figure 2B). Thermal stable proteins are attractive structural targets and we have identified crystallization conditions.

[0066] One limitation to crystallization can be intrinsic flexibility. As an alternative to screening orthologous Cascade complexes (i.e., T. thermophilus), limited proteolysis and mass spectrometry analysis may be used to identify regions susceptible to protease trimming. Limited proteolysis is routinely used to establish stable core complexes that are often more amenable to crystallization, and combining this technique with denaturing gel electrophoresis and electrospray mass spectrometry can reveal the identity of flexible regions on the surface of the complex. Our data suggest that Cascade from E. coli is amenable to structure determination using this technique.

Example 3. e ulation of expression of specific genes sssisig programmable Cascade [0067] In this Example, synthetic CRISPR/cas complexes were generated and tested to determine if Cascade can be used as a programmable delivery system for repression of gene expression.

[0068] A series of synthetic CRISPR/cas loci that contain one or more spacer sequences complementary to specific regions of a reporter gene (i.e., green fluorescent protein; "GFP") were designed. Spacer sequences were designed to target either the coding or the template stand of the target gene by annealing overlapping oligonucleotides followed by ligation and cloning. Each spacer sequence of the CRISPR was designed to target regions of the GFP sequence with a TT motif 3' of the protospacer (Figure 3A). To determine if Cascade can be used for temporal repression of GFP expression in a sequence-specific fashion and in the absence of target cell destruction, co-transformed E. coli BL21 DE3 cells were co-transfected with a streptomycin resistant plasmid containing the Cascade gene cassette (i.e., casA-E; other Cas proteins, including Cas3, were not included in the synthetic CRISPR/cas loci), one of several different GFP-targeting synthetic CRISPR/cas loci on a Kanamycin resistant plasmid and an Ampicillin resistant GFP expression plasmid (i.e., pGLO) under the control of an arabinose inducible pBAD promoter. Cascade genes and CRISPR RNA expression were induced with IPTG, and GFP expression was induced with L-arabinose (Figure 3B). Visual inspection of cell pellets indicated that only cells containing an anti-GFP CRISPR repressed GFP expression in arabinose induced cells (Figure 3B, pGLO/Cascade/anti-GFP +L-arabinose + IPTG, bottom right panel). IPTG and arabinose levels were optimized and quantification of GFP repression was performed for 8 different CRISPR constructs (4 that target the template stand and 4 that target the coding strand) and a non-GFP targeting CRISPR control (i.e. anti -phage CRISPR). Quantitative analysis of GFP expression was automated using a high-performance multi-mode plate reader (Synergy 2, BioTek). Cells were cultured in a 96-well plate agitated at 220 rpms in a chamber maintained at 37°C. Shaking stopped every 15 mins for a total of 30 seconds while the optical density of each well was measured at 600 nm and then GPF expression was assessed by excitation at 395 nm and emission was recorded at 508 nm. Cells transformed with three plasmids were cultured in media with thee antibiotics (i.e.. Step, Kan, and Amp) and these cells exhibited a protected lag -phase compared to cells that only contained the pGLO (pGLO control) plasmid (Amp only). Strains expressing GFP, Cascade and a control (anti-phage) CRISPR started to express GFP at approximately the same time as the stains containing anti-GFP CRISPRs, but GFP expressio in the control (anti-phage) strain saturated the detector ~·12 hrs after inoculation, whereas GFP expression in the cells containing anti-GFP CRISPRs plateaued at ~16 hrs (Figure 3C). Furthermore, the sequence specific reduction in GFP signal correlated with the increase of anti- GFP spacer sequences in the CRISPR (Figure 3C, black arrows).

[0069] Figure 4 shows colonies of E. coli cells transfected with control (non-GFP) plasmid, pGLO plasmid, or pGLG plasniid with GFP-targeting CRISPR and cascade cassette. GFP+ colonies of E. coli cells were evident in pGLOgroups (middle row), but were not evident in control (no pGLO) groups or groups transfected with the pGLO plasmid and GFP-targeting CRISPR. and Cascade cassette (bottom, row). The results of the study indicated that synthetic CRISPR/cas loci can be utilized to target specific genes for repression.

Example 4. Engineering Cascade to reguiate gene expression

[0070] In this Example, the amenability of genes to regulation using the synthetic

CRISPR Cascade system is further demonstrated. In addition, the use of Cascade to silence a single gene or multiple genes simultaneously, and the use of Cascade to tune expression of the target gene(s) by adjusting the target site location (i.e. blocking recognition of the transcriptional promoter to serve as a potent inhibitor) and/or by designing spacers with that are imperfectly complementary (i.e., less than 100% complementary) to the target sequence is demonstrated. Cas3 is not, included in the experiments, and thus the results of the study will show that gene expression can be repressed without cleaving of target genes via Cas nucleases. The results of the study will further show that repression of gene expression is tunable based on number of target sites, target site location, and extent that the spacer sequences are complementary to the target sequences.

[0071] Synthetic CRISPR-mediated regulation of GFP encoded on a plasmid is described herein. However, it is noted that the compositions and methods described herein can be applied to any gene of interest and can be applied to genes on any prokaryotic or eukaryotic genome. Total RNA is isolated from cells containing synthetic CRISPRs that target a single site or multiple sites on a gene of interest. For example, cells contain synthetic CRISPRs that target a single site on GFP (e.g., position 1 , 2, 3, or 4 as shown in Fignre 3A), or multiple sites on GFP

->7 (e.g., positions 1 , 2, 3, and 4 as shown in Figure 3A), In addition, for example, cells contain synthetic CRISPRs that comprise sequences that are not 100% complementary to positions 1 , 2,

3, and/or 4 of GFP. Cells containing a control synthetic CRISPR that does not target the gene of interest are also used. Equal amounts of total RNA from each ceil line are separated on 2% agarose gels containing formaldehyde, transferred to nitrocellulose membranes, and subjected to Northern blot analysis using^3zP-labeled probes generated by random hexamer amplification of the gene of interest, for example, GFP (Ready-to-go-DNA Labeling beads, GE Healthcare). Hybridization, detection, and quantification of transcripts are performed using standard methods that are well known by those having ordinary skill in the art and are described in Sambrook, J. et a!., (1989) Molecular Cloning: A. Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0072] Northern blot analysis reveals short transcripts of GFP corresponding to the distance between the transcriptional start site and the target site specified by each crRNA, (Figure 3A -300, 400, 500 and 600 nucleotides), as well as full-length GFP (-730 nucleotides). The intensity of the full-length GFP band correlates with GFP expression patterns in the plate reader assay described above in Example 5,

[0073] The results of the study indicate that Cascade competes with transcriptional initiators and/or blocks elongation of RNA polymerase on genes specified by spacer sequences engineered into the synthetic CRJSPR/cas locus, and that the extent of target gene repression may be manipulated using particular target locations, numbers of targets, and extent of that the spacer sequences are complementary to the target sequence.

Example 5. Engineering syssthetic CRISPRs for turning on gene expression

[0074] A series of synthetic CRISPRs that target genes encoding non-essential heat shock proteins have been designed. Six CRISPRs that each target a different heat shock gene have been synthesized using the method described above. In addition, CRISPRs that target 2, 3,

4, 5 or all 6 genes simultaneously have been designed.

[0075] In this Example, the CRISPRs targeting one or more heat shock gene are used to demonstrate that Cascade can be engineered to deliver transcriptional initiation factors to the promoters of specific genes. Sigma factor 32 (o^{J ~}) is genetically tethered to the C terminus of

CasA or the N terminus of CasE. a^j is engineered to dangle from the end of Cascade in a way that will permit DNA binding and RNA polymerase recruitment without steric clashing. These regions are selected by docking high-resolution crystal structures of CasA and CasE into the cryo-E map of Cascade, These two proteins are located at opposing ends of the Cascade complex and biochemical studies have shown that fusions at these locations do not perturb Cascade assembly. Similarly, the high-resolution structure of the transcriptional initiation complex from T. thermophilus, which includes the RNA polymerase, σΑ, and a fragment of the promoter DNA, provides a molecular blueprint for predicting how o'² might interact with promoter DNA and the RNA polymerase in the context of a Cascade fusion. Using these structures to guide the design, synthetic CRISPRs that target a sequence adjacent to promoters recognized by σ^'" are designed.

[0076] Cells expressing a CRlSPR/cas locus targeting one or more genes are co- expressed in E. coli BL21 DE cells, with or without, the Cascade σ³² fusion constructs.

Expression of Cascade and CRISPR RNA is induced with 1FTG when cells reach an optical density (OD600) of 0.5. Cells are maintained at 37°C until they hit an OD600 of ~1.0, at which point each of the cultures is split. One half remains at 37°C, while the other is incubated at 42°C for 15 min. These ceils, as well as control cultures that do not contain CRISPRs or cas genes, are harvested by centrifugation and total RNA is isolated for Northern blot analysis using probes against each of the heat shock genes. Quantitative PCR is also performed.

[0077] These data demonstrate temporal control of specific heat shock proteins in E, coli using IPTG rather than heat shock and demonstrate that expression will occur from <f² promoters with adjacent crRNA binding sites, but not from other a^j promoters. Thus, the results of the study will show that Cascade is a programmable complex that can be used to activate gene expression by delivering transcriptional initiation factors to specific promoters. Example 6. Anti-CRISPR proteis binds the Cascade-like complex

[0078] Virally-encoded suppressors of CRISPRs (e.g., anti-CRISPR proteins) are described herein as a method for controlling synthetic CRISPR-mediated modulation of gene expression. The Cascade-like complex, sometimes referred to as the Csy-complex, was incubated with an anti-CRISPR, and the bound (Csy-complex + anti-CRISPR) or unbound (anti-CRISPR alone) complexes were purified. As shown in Figure 5, the anti-CRISPR bound to the Cascadelike protein, indicating that an anti-CRISPR protein may be used to regulate Cascade-like synthetic CRISPR-mediated gene modulation.

[0079] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0080] The foregoing detailed description has been given for clarity of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0082] While the mvention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. References

All references cited anywhere in this specification including those cited anywhere above, are incorporated herein by reference in their entirety and for all purposes.

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Claims

1. A method of modulating the expression or function of one or more target DNA sequences in a cell comprising: expressing a synthetic CRJSPR/cas complex in a cell comprising the one or more target DNA sequences, wherein the CRISPR/cas complex comprises a CR1SPR locus comprising one or more spacer sequences complementary to the one or more target DNA sequences, wherein the spacer sequences hybridize to said one or more target DNA sequences, and wherein said one or more target, DNA sequences are not cleaved or degraded,

2. The method of claim 1, wherein the CRISPR/cas complex comprises Cascade or a Cascadelike complex.

3. The method claim 2, wherein the Cascade or Cascade-like complex is selected from the group consisting of Cascade, aCASCADE, the Csy-complex, and I-C Dvulg Cascade.

4. The method of claim 1, wherein the CRISPR'cas complex is capable of multiplexed modulation of target DNA sequences.

5. The method of claim 1, wherein the CRISPR'cas complex does not comprise a nuclease.

6. The method of claim I, wherein said one or more target DNA sequences are selected from the group consisting of a promoter sequence, an enhancer sequence, and a repressor sequence.

7. The method of claim 1 , wherein expression of said one or more target DNA sequences is reduced.

8. The method of claim 7, wherein expression of said one or more target DNA sequences is reduced by at least 10%.

9. The method of claim 1 , wherein expression of said one or more target DNA sequence is increased.

10. The method of claim 9, wherein expression of said one or more target DNA sequences is increased by at least 10%.

1 1. The method of claim 1 , wherein the CRISPR/cas complex comprises one or more proteins that are tethered to a transcriptional initiation factor.

12. The method of claim 1 1 , wherein the transcriptional initiation factor is tethered to the C- terminus or the N-terminus of the one or more proteins,

13. The method of claim 1 1 , wherein the transcriptional initiation factor is tethered to CasA or CasE.

14. The method of claim 13, wherein the transcriptional initiation factor is tethered to the C- terminus of CasA.

15. The method of claim 13, wherein the transcriptional initiation factor is tethered to the N- terminus of CasE.

56. The method of claim 1 1 , wherein the transcriptional initiation factor is a sigma factor.

17. The method of claim 16, wherein the transcriptional initiation factor is O³².

18. The method of claim 1 , wherein the CRISPR/cas complex further comprises a nuclear localization signal.

19. The method of claim 1 , wherein the CRISPR/cas locus induces at least 85% fewer off-target effects as compared to a method utilizing TALENs and/or Zinc finger nucleases.

20. The method of claim 1 , wherein the method does not induce off-target effects.

21. The method of claim 1 , wherein the spacer sequence is at least 85% complementary to the target DNA sequence.

22. The method of claim 1 , wherein the spacer sequence is at least 95% complementary to the targeted DNA sequence.

23. The method of claim 1 , wherei the spacer sequence is 100% complementary to the target DNA sequence.

24. The method of claim 1 , wherein the cell is a prokaryotic cell.

25. The method of claim 1 , wherein the cell is a eiikaryotic cell.

26. The method of claim 25, wherein the eukaryotic cell is present in an animal.

27. The method of claim 26, wherein the animal is a human.

28. The method of claim 1 , wherein the synthetic CRISPR/cas complex comprises more than one spacer sequence, and wherein the spacer sequences hybridize to different DNA target sequences.

29. The method of claim 28, wherein the DNA target sequences are within the same gene

30. The method of claim 28, wherein the DNA target sequences are within different genes,

31. The method of claim 30, wherein the different genes are in the same biological pathway.

32. The method of claim 30, wherein the different genes are in different biological pathways.

33. The method of claim 1 , wherein the method further comprises the use of one or more suppressors of CRISPR.

34. The method of claim 33, wherein the suppressor of CRISPR alters the modulation of the target DNA sequence by the synthetic CRISPR cas complex.

35. The method of claim 33, wherein the suppressor of the CRiSPR comprises one or more anti- CRiSPR proteins.

36. The method of claim 1 , wherein the method comprises expressing two or more synthetic CRISPR/cas complexes in the cell.

37. The method of claim 36, wherein the two or more synthetic CRISPR/cas complexes comprise spacer sequences that hybridize to different DNA target sequences.

38. The method of claim 37, wherein the DNA target sequences are within the same gene.

39. The method of claim 37, wherein the DNA target sequences are within different genes.

40. The method of claim 1 , wherein the method inhibits horizontal gene transfer.

41. A method of treating, preventing, or ameliorating a disease or condition caused in whole or in part by one or more target DNA sequences, said method comprising expressing a synthetic CRISPR/cas locus in the cell of an animal or plant having the disease or condition, wherein said synthetic CRISPR/cas locus comprises one or more spacer sequences complementary to said one or more target DNA sequences, wherein said spacer sequences hybridize to said one or more target DNA sequences, wherein the expression or function of said one or more target DNA sequences is modulated as a result of the hybridization to the spacer sequence, and wherein said one or more target DNA sequences are not cleaved or degraded,

42. The method of claim 41 , wherein the synthetic CRISPR/cas complex is a Cascade or Cascade-like complex.

43. The method of claim 41 , wherein multiple target DNA sequences are modulated by the synthetic CRISPR/cas complex,

44. The method of claim 41 , wherein the animal is a human.

45. A method of treating or preventing or ameliorating an infection comprising: expressing a synthetic CRISPR/cas complex in a subject infected by an infectious agent or at risk of infection by an infectious agent, wherein said synthetic CRISPR/cas complex comprises a CRISPR locus comprising one or more spacer sequences complementary to one or more target DNA sequences from said infectious agent, wherein said spacer sequences hybridize to said one or more target DNA sequences, wherein the expression or function of said one or more target DNA sequences is modulated as a result of the hybridization to the spacer sequence, and wherein said one or more target DNA sequences are not cleaved or degraded.

46. The method of claim 45, wherein the synthetic CRISPR/cas complex is a Cascade or Cascade-like complex.

47. The method of claim 45, wherein multiple target DNA sequences are modulated by the synthetic CRISPR/cas complex. 48, The method of claim 45, wherein the infectious agent is a bacterium.

49. The method of claim 45, wherein the infectious agent is a virus. 50 The method of claim 45, wherein the infectious agent is a fungus.

51. The method of claim 45, wherein the subject is an animal or plant.

52. The method of claim 45, wherein the subject is a human.

53. The method of claim 2, wherein the Cascade complex comprises one or more proteins comprising an amino acid sequence according to SEQ ID NO: 1 , 2, 3, 4, or 5 or fragments thereof.

54. A. method of modulating a target RNA sequence in a cell comprising; expressing a Cascade or Cascade-like synthetic CRISPR/cas complex in a cell comprising the one or more target RNA sequences, wherein the CRISPR is specific for the target RNA sequence, wherein the Cascade or the Cascade-like complex comprises an RNase, and the RNA target is degraded.