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Title:
CRISPR (CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS) RNA-GUIDED CONTROL OF GENE REGULATION
Document Type and Number:
WIPO Patent Application WO/2014/093479
Kind Code:
A1
Abstract:
Methods, systems, and compositions for programmable gene modulation based on clustered regularly interspaced short palindromic repeats (CRISPRs) are provided. The methods comprise expressing a synthetic CRISPR/cas locus in a cell comprising one or more target DNA sequences for modulating the expression or function of one or more target DNA sequences in a cell, and the one or more target DNA sequences are not cleaved or degraded.

Inventors:
WIEDENHEFT BLAKE (US)
Application Number:
PCT/US2013/074372
Publication Date:
June 19, 2014
Filing Date:
December 11, 2013
Export Citation:
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Assignee:
UNIV MONTANA STATE (US)
International Classes:
A61K48/00; C12N1/20
Domestic Patent References:
WO2010075424A22010-07-01
Foreign References:
US20100076057A12010-03-25
US20100192237A12010-07-29
Other References:
SINKUNAS ET AL.: "Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.", EMBO J., vol. 30, no. 7, 2011, pages 1335 - 1342
KOURENNAIA ET AL.: "Mutational Analysis of Escherichia coli Heat Shock Transcription Factor Sigma 32 Reveals Similarities with Sigma 70 in Recognition of the -35 Promoter Element and Differences in Promoter DNA Melting and -10 Recognition.", J BACTERIOL., vol. 187, no. 19, 2005, pages 6762 - 6769
Attorney, Agent or Firm:
VEITENHEIMER, Erich et al. (1299 Pennsylvania Avenue N.W.,Suite 70, Washington District of Columbia, US)
Download PDF:
Claims:
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.

Description:
CRISPR (CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS) RNA-GUIDED CONTROL OF GENE REGULATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Nos. 61/735,876, filed December 1 1 , 2012, and 61/799,531, filed March 15, 2013, the contents of each of which are herein incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under P20GM103500 awarded by the National Institutes of Health. The government has certain rights in the invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0003] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety; A computer readable format copy of the Sequence Listing

(filename: MONTJ40_01WO_SeqList_ST25.txt, date recorded: December 10, 2013, file size 7 kilobytes).

FIELD OF THE INVENTION

[0004] The invention relates to programmable gene modulating systems that offer a novel approach for systemically controlling the expression of a single gene or dozens of genes simultaneously.

BACKGROUND OF THE INVENTION

[0005] Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of a recently discovered, nucleic-acid-based adaptive immune system that is widespread in bacteria and archaea and serves as protection against phages and other invading nucleic acids. [0006] Each CRISPR/cas locus is comprised of a series of direct repeats that are separated by nonrepetitive spacer sequences derived from foreign genetic elements. An a denine - and thymiiie-rich leader sequence that contains a transcriptional promoter is located at one end of the locus. In addition to the leader sequence, comparative analyses have also identified a variable cassette of CRISPR-associated (cas) genes, which is typically located adjacent to a CRISPR/cas locus. The Cas proteins encoded by these genes are required for new sequence acquisition, CRISPR RNA biogenesis and target sequence identification and destruction. CRISPR/cas loci are transcribed and the long primary transcript is processed into a library of short CRJSPR-derived RNAs (crRNAs), also known as spacer sequences, that contain a unique sequence complementary to a foreign nucleic acid challenger. In Escherichia coli K12, CRISPR/cas loci are transcribed and processed into small RNAs that are incorporated into a multi-subumt surveillance complex called Cascade (CRISPR-associated complex for antiviral defense), which is required for protection against bacteriophages via high affinity binding to DNA targets that contain a sequence complementary to the crRNA-guide (spacer sequences). Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) that is required for target degradation. In other CRISPR systems, target DNA is degraded by Cas9, or other dedicated nucleases, CRISPR systems are in turn regulated by suppressors of CRISPR systems, such as anti-CRISPR proteins, which are encoded by some bacteriophages and which inactivate the CRISPR system, thereby evading the bacterial system for surveillance and targeting of foreign DNA.

[0007] RN A interference (RNAi) is a posttranscriptional gene regulation mechanism that relies on small RNAs as guide for target sequence identification. In eukaryotes, long double- stranded RNAs (dsR As) are processed into 21-nt small interfering RNAs (siRNAs) by a dedicated RNase III enzyme called Dicer. One strand of the siRNA is loaded into an Argonaute protein (AGO)-containing regulatory complex, where the siRNA serves as a sequence-specific guide that delivers the AGO nuclease to invading viral nucleic acids. The RNAi machinery has been exploited as a method for silencing gene expression in a cell for research applications including determining gene functions, as well as potential therapeutic applications. However, such systems are limited by difficulties in designing effective RNAi sequences; problems with identifying safe and efficient methods of delivery of the RNAi sequence to the target cell; and non-specific or off-target effects of the RNAi system. Such issues have limited the use of RNAi systems, particularly with regard to therapeutic applications. Other technologies for gene regulation such as TALENs (Trans activator-like endonucleases) and Zinc finger nucleases are associated with a high degree of off-target effects. Moreover, none of these technologies can be used to positively modulate, or turn on, gene expression.

[0008] The technology provided herein is mechanistically distinct from eukaryotic

RNAi. The technology provided herein is also distinct from, the recently described systems for genetic modulation utilizing Cas9 CRISPR. systems. There is a need to provide simple, inexpensive, safe, versatile, and effective means to turn off or turn on gene expression and function in a programmable fashion.

SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention provides a method of modulating the expression or function of one or more target DNA. or R.NA sequences in a cell. In a further embodiment, the one or more target DNA sequences are not cleaved or degraded. In a further embodiment, the method comprises expressing a synthetic CRISPR/cas locus in a cell comprising one or more target DNA sequences, wherein the CRISPR. comprises one or more spacer sequences complementary to the one or more target DNA sequences, and wherein the spacer sequences hybridize to said one or more target DNA sequences. In one embodiment, the synthetic CRISPR/cas system is a Cascade or a Cascade-like CRISPR/cas locus. In one embodiment, the CRISPR/cas system is capable of multiplexed modulation of target DNA sequences. Thus, in one embodiment, the method comprises expressing a synthetic CRISPR/cas system in a ceil comprising two or more target DNA sequences, wherein the CRISPR comprises two or more spacer sequences complementary to the two or more target DNA sequences, and wherein the spacer sequences hybridize to said two or more target DNA sequences and modulate expression or function of said target DNA sequences. In a further embodiment, the method comprises modulating expression or function of multiple target sequences, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or more target DNA sequences. In one embodiment, the multiple target sequences are modulated by expression of a single CRISPR/cas locus in the cell. [0010] In one embodiment, expression of one or more target DNA sequences is diminished. In another embodiment, the expression of one or more target DNA sequences is increased. In one embodiment, the method provided turns on a gene that was not previously expressed. In one embodiment, the CRISPR/cas system is a Cascade or Cascade-like system. In one embodiment, the Cascade or Cascade-like system does not comprise an active nuclease. In a further embodiment, the Cascade or Cascade-like system does not comprise Cas3.

[0011] In one embodiment, the CRISPR/cas complex is any type of CRISPR/cas complex. In one embodiment, the CRISPR/cas complex is a Cascade-like CRISPR/cas complex. Cascade-like CRISPR/cas complexes include, but are not limited to, Cascade, aCASCADE, the Csy-complex, and I-C Dvulg Cascade (see, for example, Bourns et al. Science (2008);321 (5891):960-4; lore et al Nat Struct Mo! Biol 2011; 18(5) :529-36; Wiedenheft et al Nature 2011 ;477(7365):486-9; Lintner et al J Biol Chem. (201 1) 286(24):21643-56; Wiedenheft et al N452011 ;108(25): 10092-7; and Nam et al., Structure 20122012 Sep 5;20(9): 5574-84). In another embodiment, the CRISPR/cas complex is a Type I CRISPR/cas complex. In a yet further embodiment, the Type 1 CRISPR/cas complex is a CR1SPR Cascade complex. In another embodiment, the CRISPR/cas system is a Type II CRISPR/cas system In another embodiment, the CRISPR/cas system is a Type III CRISPR/cas system.

[0012] In one embodiment, the CRISPR/cas system is expressed in a cell and the Cas proteins and crRNA are expressed and self-assemble into a CR1SPR complex capable of binding to target DNA sequences and recruiting Cas nucleases for target DNA sequence destruction. In one embodiment, the components of the CRISPR/cas system are introduced into the ceil on separate expression vectors. In another embodiment, the components of the CRISPR/cas system are introduced into the cell on a single expression vector.

[0013] In one embodiment, the present invention provides a method of modulating the expression of one or more target DNA sequences comprising expressing a synthetic CRISPR'cas locus comprising spacer sequences complementary to said one or more target DNA sequences to a cell, wherein the spacer sequences hybridize to said one or more target DNA sequences, and wherein expression of said one or more target DNA sequences is turned on or increased. In a further embodiment, multiple target DNA sequences are turned on or increased. In one embodiment, one or more of the Cas proteins is tethered to a transcriptional initiation factor. Thus, in one embodiment, the CRISPR/cas system directs expression of the target sequence. In a further embodiment, the protein that is tethered to a transcriptional initiation factor is CasA, and the transcriptional initiation factor is tethered to the C-terminus of CasA. In another embodiment, the protein that is tethered to a transcriptional initiation factor is CasE, and the transcriptional initiation factor is tethered to the N-terminus of CasE. In another embodiment, the transcriptional initiation factor is a sigma factor. In a further embodiment, the transcriptional initiation factor is sigma 32 (σ 32 ). In another embodiment, the transcriptional initiation factor is a eukaiyotic transcriptional initiation factor, Eukaiyotic transcription factors are any factors that can activate or repress transcription of a single eukaryotic gene or a number of eukaryotic genes, and include DNA binding proteins, proteins that bind DNA binding proteins, protein kinases, protein phosphatases, protein methyltransferases, GTP-binding proteins, and the like. Such transcription factors are well known in the art and include, but are not limited to, STAT family proteins (e.g., STATs 1 , 2, 3, 4, 5, and 6), fos/jun, NFkappaB, HIV-Tat, and the E2F family.

[0014] In one embodiment, the present invention provides a method of modulating the expression of a target DNA sequence comprising expressing a synthetic CRISPR/cas locus in a eukaiyotic cell comprising a target DNA sequence. In a further embodiment, the CRISPR/cas locus is codon optimized for eukaiyotic expression. In a further embodiment, the CRISPR/cas locus further comprises a nuclear localization signal.

[0015] In one embodiment, a method is provided for modulating the expression of one or more target DNA sequences comprising expressing a synthetic CRISPR/cas locus in a cell comprising said one or more target DNA sequences, wherein the method provided induces fewer off-target effects as compared to other methods known in the art, such as TALENs and Zinc finger nucleases. In a further embodiment, the method induces at least 80% fewer off-target effects as compared to other methods of gene targeting known in the art. In a yet further embodiment, the method provided induces at least 100% fewer off-target effects as compared to a. other methods of gene targeting known in the art. In another embodiment, the method provided does not induce off-target effects.

[0016] In one embodiment, the present invention provides a method of modulating the expression or function of a target DNA sequence in a cell wherein the target DNA sequence is not cleaved or degraded, comprising expressing a synthetic CRISPR/cas system in a cell comprising a target DNA sequence, wherein said CRISPR comprises one or more spacer sequences complementary to the target DNA sequence, wherein said spacer sequences hybridize to said target DNA sequence, and wherein the spacer sequence is at least 85% complementary to the target DNA sequence. In a further embodiment, the spacer sequence is at least 95% complementary to the target sequence. In a yet further embodiment, the spacer sequence is 100% complementary to the target DNA. sequence. Thus, in some aspects, transcriptional repression or activation of the target gene is tunable based on the extent that the spacer sequence is complementary to the target sequence. For example, a spacer sequence that is less than 100% complementary to the target sequence may repress or activate expression of a DNA. sequence to a lesser extent compared to a spacer sequence that is 100% complementary to the target sequence.

[0017] In one embodiment, the synthetic CRISPR/cas locus is specific for more than one site on a target gene. In another embodiment, the number of sites within a gene that are targeted may affect the extent of repression or activation of a target gene. For example, a CRISPR may comprise spacer sequences complementary to 1 , 2, 3, 4, 5, 6, 7, 8 or more sites on either the coding or template stand of a gene or in a promoter region of the gene. In a further embodiment, a CRISPR comprising spacer sequences complementary to multiple genes affects repression or activation of a target gene to a larger extent than a CRISPR comprising spacer sequences complementary to one site on a gene.

[0018] In one embodiment, the synthetic CRISPR/cas system can be used to regulate gene expression by targeting a promoter, enhancer of gene expression, repressor of gene expression, or any other sequence involved in the control of gene expression. In one embodiment, the synthetic CRISPR/cas locus is specific for a promoter sequence. In another embodiment, the synthetic CRISPR/cas locus is specific for an enhancer or repressor of gene expression.

[0019] In one embodiment, the methods provided further comprise the use of suppressors of CRISPRs to reverse the synthetic CRISPR-mediated gene regulation. Suppressors of CRISPR may be anti-CRISPR proteins, including virally-encoded proteins, non-coding RNA, or other materials that interfere with the CRISPR system. Thus, in one embodiment, the synthetic CRISPR/cas system may be used to turn on or turn off gene expression, and the effect of the synthetic CRISPR/cas system may be immediately reversed through the expressions of one or more suppressors of CRISPR in the cell. In one embodiment, anti-CRISPR proteins bind directly to the Cascade or Cascade-like complex, thereby interfering with gene regulation.

[0020] Thus, in one embodiment, the compositions and methods provided herein provide a programmable system, for modulation of gene expression that includes the ability to turn on as well as turn off genes; allows modulation that may be multiplexed; is associated with few or no off-target effects; is tunable based on the number of targeted sites on a gene or the location of the targeted sites (e.g., promoter targets); and is reversible via suppressors of CRISPR.

[0021] In one embodiment, the method comprises expressing two or more synthetic

CRISPR/cas loci in a cell. In a further embodiment, the two or more synthetic CRISPR/cas loci comprise spacer sequences that hybridize to different DNA target sequences. In a further embodiment, the DNA target sequences are in the same biological pathway. In a further embodiment, the DNA target sequences are in different biological, pathways. In one embodiment, the present in vention pro vides a method of modulating the expression or function of one or more target DNA sequences in a cell wherein the cell is a prokaryotic cell, bacterial or archaeal. A target DNA may be dsDNA or ssDNA. In one embodiment, the present invention provides a method of modulating the expression or function of one or more ssRNA target. In one embodiment, a method for modulating one or more target RNA sequence in a cell is provided, comprising expressing a Cascade or Cascade-like 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 RN A target is degraded. In one embodiment, the target RNA sequence is a microRNA.

[0022] In another embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is present in a subject. In another embodiment, the subject has a disease or condition caused in whole or in part by the one or more target DNA sequences. In another embodiment, the cell is a. eukaryotic cell, and the cell is present in a. subject. In a further embodiment, the subject is a human.

[0023] In one embodiment, the present invention provides a method of treating, affecting, or ameliorating a disease or condition caused in whole or in part by one or more target DNA sequences comprising expressing a synthetic CRISPR/cas complex in the cell of an animal or plant having the disease or condition, wherein said synthetic CRISPR/cas complex comprises a one or more CRISPR comprising one or more spacer sequences complementary to said one or more target DNA sequences, and wherein the spacer sequences hybridize to the one or more target DNA sequences. In a further embodiment, the synthetic CRISPR/cas complex is a Cascade or Cascade-like complex. In a further embodiment, the activity, expression, or function of one or more target DNA sequences are modulated as a result of the hybridization to the spacer sequences. In a further embodiment, the activity, expression, or function of multiple target DNA sequences associated with the disease or condition are modulated by the synthetic CRISPR/cas complex. In a further embodiment, the one or more target DNA sequences are not, cleaved or degraded. In a further embodiment, the animal is a human. In another embodiment, the animal is a livestock animal, such as, for example, a pig or a cow. In another embodiment, the plant is a plant grown for agricultural purposes. In an additional embodiment, the present invention provides a method of treating, preventing, or ameliorating a disease or condition caused in whole or in part by one or more target DNA and/or RNA sequences comprising expressing a synthetic CRISPR/cas complex in the cell of an animal or plant, wherein said synthetic CRISPR/cas complex comprises one or more spacer sequences complementary to one or more target DNA and/or RNA sequences, and wherein the spacer sequences hybridize to said one or more target DNA and/or RNA sequences. In a further embodiment, the synthetic CRISPR/cas complex is a Cascade or Cascade-like complex. In a further embodiment, the activity, expression or function of one or more target DNA and/or RNA sequences are modulated as a result of the hybridization to the spacer sequence. In a further embodiment, the activity, expression or function of multiple target DNA and/or RNA sequences are modulated by the synthetic CRISPR/cas complex. In a further embodiment, the animal is a human. In another embodiment, the animal is a livestock animal, such as, for example, a pig or a cow. In another embodiment, the plant is a plant grown for agricultural purposes.

[0024] In one embodiment, the present invention provides a method of treating, preventing, or ameliorating an infection comprising expressing a synthetic CRISPR'cas locus in the cell of a subject infected by an infectious agent or at risk of infection by an infectious agent, wherein said synthetic CRISPR/cas locus comprises one or more spacer sequences complementary to one or more target DNA and/or R A seque ces from said infectious agent, and wherein the spacer sequences hybridize to said one or more target DNA and/or RNA sequences. In a. further embodiment, the synthetic CRISPR/cas locus is a. Cascade or Cascadelike complex. In a further embodiment, the one or more target DNA and/or RNA sequences are modulated as a result of the hybridization to the spacer sequence. In a further embodiment, multiple target DNA and/or RNA sequences from the infectious agent are modulated by the synthetic CRISPR/cas locus. In a further embodiment, the target DNA sequences are not cleaved or degraded. In a further embodiment, the infectious agent is a bacterium, virus, fungus, LINEs (Long interspersed elements); SINEs (Short, interspersed elements) or other genetic parasite. In another embodiment, the subject is an animal, for example, a human. In another embodiment, the subject is a plant or plant cell .

[0025] In one embodiment, the present invention provides a method of inhibiting horizontal gene transfer. Horizontal gene transfer includes, but is not limited to, phage transduction, DNA transformation, and plasmid conjugation. In one embodiment a method is provided in which CRISPR/cas complex are expressed in a cell, tissue, or subject in order to inhibit horizontal gene transfer into the DNA of said cell, tissue, or subject. In some embodiments, the subject is an arc ea or bacteria.

BRIEF DESCRIPTION OF THE FIGURES

[0026] Figure 1 shows Cascade protein subunits (CasA, CasB, CasC, CasD, and CasE) and crRNA following expression and purification of the self-assembling complex from E. coli cells.

[0027] Figure 2A shows purification of the holo Cascade, or subcomplexes that are missing either CasA (-A) or CasA and CasB (-B). Figure 2B show's the purified Cascade complex from T, thermophilics.

[0028] Figure 3A depicts the regions of the GFP sequence for which complementary spacer sequences were generated (i.e., region 1 , 2, 3, and 4) to design synthetic CRISPR/cas loci that contain one or more spacer sequences (i.e., CRISPR/cas loci that contain 1 , 2, 3, or 4 complementary spacer sequences). Figure 3B shows cell pellets from cells expressing the GFP expression plasmid (pGLO) alone (top row): cell pellets from cells expressing the pGLO plasmid and an anti-phage CRISPR (middle row); and cell pellets from cells expressing the pGLO plasmid and the anti-GFP CRISPR with the Cascade gene cassette (i.e., casA-E) (bottom row), in the presence of IPTG (induces Cascade and CRISPR) alone (left column); L-arabinose (induces GFP expression) alone (middle column); or both IPTG and L-arabinose (right column), Figsire 3C depicts a quantitative analysis of GFP expression in cells expressing BL21 control plasmid; pGLO control plasmid; control plasmid comprising an anti-phage CRISPR; and plasmids comprising pGLO and the indicated anti-GFP CRISPR with the Cascade gene cassette. Black arrows indicate that the sequence-specific reduction in GFP signal correlates with the number of anti-GFP spacer sequences in the CRISPR. These experiments were repeated in triplicate; error bars represent one standard deviation.

[0029] Figure 4 shows white light images (left column of images) as well as UV-light images (right column of images) of representative colonies of the E, coli cells from GFP- CRJSPR studies. In the right column of images, GFP+ or GFP- (green or not green, respectively) colonies of E. coli cells alone (no pGLO plasmid; top row), E. coli cells expressing the pGLO plasmid alone (middle row), or E. coli cells expressing the pGLO plasmid with GFP-targeting CRISPR and Cascade cassette (bottom row) are shown.

[0030] Figure 5 shows binding of an anti-CRISPR protein to the Cascade-like complex

Csy-complex.

DETAILED DESCRIPTION

[0031] Viruses that infect bacteria (i.e., bacteriophages) are the most abundant and diverse biological agents on the planet. Bacteriophages and plasmids are major purveyors of traits that confer antibiotic resistance, enhance bacterial adhesion, colonization, invasion, dissemination through human tissues, resistance to immune defenses, transmissibility among humans and exotoxin production. The selective pressures imposed by bacteriophages and plasmids have a profound impact on the composition and behavior of microbial communities in every ecosystem. In response to these selective pressures microbes have evolved a sophisticated nucleic acid-based adaptive immune system called CRISPR (clustered regularly interspaced short palindromic repeat) (Al- Attar S. et al. Biol. Chem 2011 392(4) :277-89); Bhaya et al Annu Rev Genet 2012; 45:273-97; Westra et al Annu Rev Genet 2012 46:31 1-39; Wiedenheft et al Nature 2012 482(7385):331-8; and Sorek et al Annual Rev of Biochem 2013, each of which is incorporated herein by reference in its entirety). Bacteria and archaea acquire resistance to viral and piasmid challengers by integrating short fragments of foreign nucleic acids into the host chromosome at one end of the CRISPR (Barrangou et al Science 2007 3 i 5(5819): 1709-12; Cady et al J Bacterial 2012; Datsenko et al Nat Commun 2012 3:945; Pourcel et al Microbiology 2005 151(Pt 3):653; Swarts et al PloS One 2012 7(4):e35888; each of which is incorporated herein by reference in its entirety). Each CRISPR/cas locus comprises of a series of repeat sequences of approximately 24-48 nucleotides that are separated by unique 'spacer' sequences derived from foreign genetic elements, like viruses and plasmids. Each CRISPR/cas complex or system also includes CRJSPR-associated genes (cas genes) involved in surveillance for and acquisition and destruction of foreign DNA. Foreign DNA sequences selected for integration are called protospacers (i.e. origin of spacers) and in many CRISPR systems, protospacers are selected from regions of DNA that are flanked by a short sequence motif referred to as the protospacer adjacent motif (PAM) (Deveau et al J Bacterial 2008 190(4): 1390; Horvath et al J Bacterial 2008 190(4): 1401; Mojica et al Microbiology 2009 155(Pt 3):733 and Yosef et al Nucleic Acids Res 2012 40(12):5569; each of which is incorporated herein by reference in its entirety). In many Type I and Type II CRISPR systems, PAM sequences are essential for high affinity binding. CRISPR-mediated adaptive immune systems proceed according three distinct stages: acquisition of foreign DNA, CRISPR RNA biogenesis, and target interference. Although these three basic stages appear to be common to all CRISPR systems, CRISPR/cas loci and the proteins that mediate each stage of adaptive immunity are remarkably diverse.

[0032] In Escherichia coli, the CRISPR system is considered a Type 1 CRISPR system.

CRISPR/cas loci are transcribed and processed into small R As that are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense), which is required for protection against bacteriophages. Cascade is a 405- kDa- ribonucleoprotein complex composed of 11 subunits of five functionally essential Cas proteins (one CasA, two CasB, six CasC, one CasD and one CasE, corresponding to SEQ ID NOs: 1 , 2, 3, 4, and 5: Cas A-E are also known as Csel , Cse2, Cse4, Cas5e, and Cse3, respectively) and a 61- nucleotide crRNA. The cas genes for the Cascade-like complexes are related to but distinct from tliese sequences. Cascade finds and binds specific dsDNA sequences with high efficiency and affinity, in part through recognition of the Protospacer Adjacent Motif (PAM), which is a 2 to 5 sequence motif adjacent to the protospacer. The PAM is essential for high affinity dsDNA binding in the E. coli Cascade system, as well as in Type IA-F and Type IIA-B CRISPR systems. However Cascade, but not Cas9 can bind to ssDNA and ssRNA targets that do not have an adjacent PAM. In Type 1 systems, specifically Cascade, dsDNA binding results in base pairing that extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) that, is required for target degradation.

[0033] Type II CRISPR systems use a trans-encoded small RNA (tracrRNA) that pairs with the repeat fragment of the pre-erRNA, followed by degradation of the invading DNA by the nuclease Cas9. In Type III CRISPR systems, Cas6 is responsible for the processing step (i.e., the processing of foreign DNA for integration of the foreign DNA), but the crRNAs are believed to be transferred to a distinct Cas complex (called Csm in subtype 1Π-Α systems and Cmr in subtype III-B systems) for degradation of target sequences. The two subtypes of CRISPR -Cas type III systems target either DNA (subtype III- A systems) or RNA (subtype III-B systems).

[0034] The synthetic CRISPRs provided herein utilize CRISPR-associated complexes known as Cascade, or Cascade-like complexes, which comprise a multi-unit architecture. Thus, the Cascade or Cascade-like CRISPR/cas loci can be loaded with multiple spacer sequences, or crRNA guides. For example, in one embodiment, several spacer sequences are encoded in a single Cascade or Cascade-like CRISPR/cas locus; the Cascade or Cascade-like complex comprises a subunit (e.g., the endoribunuciease Cas6) that processes the CRISPR RNA and then loads each Cascade molecule with the spacer sequences. Thus, the single Cascade or Cascadelike CRISPR/cas complex is able to process and load multiple mature CRISPR RN As (crRNAs), thereby making possible a multiplexed CRISPR system for gene targeting. Thus, in one embodiment, provided herein are compositions and methods for multiplexed gene targeting. By "multiplexed gene targeting," is meant targeting more than one different target on one or more different genes. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 15, 20, 30 or more different targets on genes on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 30 genes that are targeted by spacers on a single CRISPR-'cas locus. [0035] Without wishing to be bound by theory, CRISPR is hypothesized to accelerate target identification through complex binding behaviors that involve fast association and dissociation rates with non-target sequences. It is further hypothesized that crRNA targeted to a particular sequence may be utilized to silence or induce expression of the target sequence. Silencing effects on the expression of a target sequence are due to crRNA hybridization inhibiting the binding of RNA polymerase or other transcription factors, resulting diminished or silenced gene expression without destruction of the target sequence; induced or increased expression of a target sequence may occur when a transcription initiation factor is tethered to a CRISPR protein expressed with the crRNA.

[0036] The present invention provides methods, systems, and compositions for use in modulating the expression and/or function of one or more target DNA sequences using synthetic CRISPR/cas loci. For example, the present invention provides methods, systems, and compositions for manipulating the genomes of organisms including bacterial, plant, and animal organisms. In some embodiments, the one or more target DNA sequences are in a eukaryotic ceil. In other embodiments, the one or more target DNA sequences are in a prokaryotic cell. In some embodiments, the cell is in a subject. In other embodiments, the cell is in vitro, or outside a subject. In some embodiments, the provided methods, systems, and compositions relate to interfering with horizontal gene transfer. In some embodiments, the compositions and methods of the present invention relate to the use of CRISPR to interfere with an organism's ability to receive horizontal gene transfer of genetic material or provide resistance against horizontal gene transfer from bacteria, viruses, and plasmids. In some embodiments, the methods, systems, and compositions provided herein prevent the exchange of bacterial DNA by transduction. In other embodiments, the methods, systems, and compositions provided herein protect a subject, tissue, or cell from the transfer of foreign genes. For example, the subject, tissue, or ceil may be protected from horizontal transfer of foreign genes from plasmids, bacteria, viruses, and the like via the methods, systems, and compositions provided herein. The present invention provides methods, systems, and compositions useful in biotechnology, medicine, veterinary medicine, research, commercial, industrial, and other fields.

[0037] In one aspect, the methods and compositions of the present invention provide programmable systems for targeted genome modification. In one embodiment, the programmable systems allow multiplexed gene targeting, i.e., targeting multiple genes with a. single synthetic CRISPR/cas locus. In one embodiment, the methods and compositions of the present invention provide programmable systems for targeting genome modification in organisms that lack facile mechanisms for generating gene knockouts. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have proven to be powerful tools for targeted genome modifications in mammalian cells. However, these technologies require elaborate and sometimes error prone methods for engineering proteins to recognize specific sequences. In contrast, programming CRISPR-RNA guide systems involves onl the simple rules of complementary base pairing and can be targeted to almost any region of the genome. Demonstrations of CRJSPR RNA-guided genome editing in human cells have been reported in two publications (Cong et al, 2013 Science Feb 15; 339(6 J 2I):8 J 9-23 and Mali et al., 2013 Science Feb 15; 339(6121 )823-6). Several publications describe CRJSPR system-related methods for gene targeting that employ the nuclease Cas9, which cuts double-stranded DNA in a manner similar to the mechanisms of other DNA editing tools known in the art. This Cas9- dependent technology has the potential to be easier and less expensive than previously utilized DNA editing tools, but lacks the versatility and easily programmable multiplexing capacity of the compositions and methods described herein. Moreover, none of the systems described in the recent publications provide the ability to modulate gene expression by turning genes on,

[0038| In some embodiments, the compositions and methods of the present invention are simpler and/or more programmable than the CRISPR systems previously reported in the art. For example, unlike Cas9-based systems and other CRJSPR systems reported in the art, the methods and compositions described herein can be easily manipulated to perform multiplexed gene regulation. In one embodiment, the programmable CRJSPR system described herein does not comprise a nuclease, or comprises an inactive nuclease (e.g., a nuclease that is catalyticaliy dead). In another embodiment, the programmable CRISPR system described herein comprises an inactive (e.g., catalyticaliy dead) nuclease. Thus, the methods and compositions disclosed herein do not result in degradation of target sequences. As another example, the methods and compositions described herein provide an easily manipulated tool for multiplexed gene targeting, including both turning on and turning off of specifically targeted genes. In some embodiments, the compositions and methods of the present invention use CRISPR RNA-guide complexes to block gene transcription. In yet further embodiments, the compositions and methods of the present invention may be used to modulate gene expression by turning a gene on. The compositions and methods of the present invention, in one embodiment, modulate gene expression with minimal toxicity and minimal off-target effects. ' The compositions and methods of the present invention may be applied to a broad spectrum of CRISPR pathways. Moreover, the compositions and methods described herein harness Cascade or a Cascade-like complex, and therefore allow many distinct crRNAs to be loaded in a single CRISPR/cas locus.

[0039] In one embodiment, the synthetic CRISPR functions to diminish or silence gene expression in the absence of nuclease activity. In one embodiment, the lack of nuclease activity results in a lower frequency of off-target effects. In one embodiment, a synthetic CRISPR/cas locus induces at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least, 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% fewer off-target effects when compared to an RNA interference system targeted to the same gene. In another embodiment, a synthetic CRISPR/cas locus does not induce any off-target effects.

[0040] The synthetic CRISPR/cas locus can be targeted to specific genes in order to block or augment expression of said genes. Thus, the compositions and methods herein are unlike RNAi at least in that they act at the DNA level. However, Cascade, but not Cas9, can also act on ssDNA and ssRNA. In some embodiments, the synthetic CRISPR/cas is designed to target RNA. In a further embodiment, Cascade is tethered to an RNase and an RNA is thereby specifically targeted and destroyed using the synthetic CRISPR/cas system. In one embodiment, the target RNA is a microRNA (e.g., a small non-coding RNA that functions as a post- transcriptionai regulator of gene expression).

[0041 ' | Without wishing to be bound by theory, it is hypothesized that Cascade or other

CRISPR/cas locus complexes inhibit the recruitment and'Or elongation of the RNA polymerase due to fast on-rates and slow off-rates of crRN A binding, and thereby silence expression of genes to which the CRISPR is directed via spacer sequences complementary to the target gene. Without wishing to be bound by theory, it is hypothesized that when one or more CRISPR protein is tethered to a transcription factor, the CRISPR localizes to the target gene to which it is directed via complementary spacer sequences, and initiates or augments expression of the target gene. [0042] In other recently described CRISPR-based gene targeting systems (e.g., Cas9- related systems), the knockdown of specific genes is reversible insofar as the Cas proteins and RNA guides are eventually diluted due to cellular growth. In one embodiment, the methods and compositions provided herein provide the ability to reverse the effects on target genes in a more controlled fashion, using suppressors of CRISPRs. Suppressors of CRISPRs are agents that, alter or interfere with the activity of CRISPRs. Suppressors of CRISPRs may be virally-encoded proteins, non-coding RNA, or other virus-derived materials that interfere with the CRISPR system. Suppressors of CRISPRs may also be agents that mimic the activity of virally-encoded suppressors of CRISPR. Suppressors of CRISPR include those described in the art, for example, in Bondy-Denomy, Nature 2013 493(7432):429-32, which is herein incorporated by reference in its entirety. Thus, in one embodiment, the synthetic CRISPR/cas locus may be used to turn on or turn off gene expression, and the effect of the synthetic CRISPR cas locus may be immediately reversed through the use of one or more suppressors of CRISPR. In one embodiment, a suppressor of the CRISPR system is an anti-CRISPR protein. In one embodiment, the anti- CRISPR protein binds directly to the Cascade or Cascade-like complex, thereby interfering with target binding, regulation, or nuclease recruitment. The skilled artisan will recognize that any suppressor of CRISPR may be used in the methods described herein to turn off the synthetic Cascade-like CRISPR system-mediated gene regulation. For example, an anti-CRISPR protein that binds to a Cascade or Cascade-like complex or component of the complex may be used in the methods described herein to turn off the synthetic Cascade of Cascade-like CRISPR- mediated gene regulation system.

[0043] In one embodiment, the CRISPR/cas system or complex is any type of

CRISPR/cas locus. In particular embodiments, the CRISPR/cas complex is a Cascade or Cascade-like complex. In one embodiment, the CRISPR/cas complex is a Type I CRISPR/cas complex. In a further embodiment, the Type I CRiSPR'cas complex is a CRISPR Cascade complex. In another embodiment, the CRISPR/cas system is a Type II CRiSPR'cas system. In another embodiment, the CRISPR/cas locus is a Type III CRiSPR'cas locus.

[0044] In one embodiment, the synthetic CRISPR/cas complex is Cascade or a Cascadelike complex such as, for example, Cascade, aCASCADE, the Csy-complex, or I-C/Dvulg Cascade. In one embodiment, the CRISPR/cas complex comprises the Cascade complex. In a further embodiment, the Cascade complex is the holo Cascade complex from a bacterial organism. In another embodiment, the Cascade complex is missing one or more Cas proteins that are normally found in the Cascade complex. For example, in embodiments, the Cascade complex is missing CasA (-A), CasB (-B), Ca.sC (-C), CasD (-D), CasE (-E), or any combination of Cas proteins (e.g., CasA. and CasB (-A-B)).

[0045] In one embodiment, the Cascade complex is the Cascade complex or a Cascade- like complex from, any bacterial organism. In one embodiment, the Cascade complex is the Cascade complex from. Escherichia coli. In another embodiment, the Cascade complex is the Cascade complex from Streptococcus thermophilus or Thermus therniophilus.

[0046] The term, "gene," as used herein, refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. The nucleic acid may also optionally include non-coding sequences such as promoter or enhancer sequences. The term "intron" refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons. As used herein, the term "genome" refers to the DNA of an organism or the RNA of a virus. The terms "genome" and "genomic DNA" encompass genetic materia! that may have undergone amplification, purification, or fragmentation. The genome may encompass the entirety of the genetic materia! from of an organism, or it may encompass one chromosome from an organism with a plurality of chromosomes.

[0047] As used herein, "nucleic acid" or "nucleic acid molecule" refers to any DNA or

RNA molecule. In some embodiments, the DNA or RNA molecule is single stranded (i.e., ssDNA or ssRNA). in other embodiments, the DNA or RNA molecule is double stranded (i.e., dsDNA or dsR A). The term "isolated nucleic acid," as used herein in connection with a DNA molecule, refers to DNA that is free of flanking genes present in the naturally-occurring genome of the organism from which the DNA is derived. The term "isolated nucleic acid" as used herein in connection with an RNA molecule refers to an mR A molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components such as other types of RNA molecules or proteins. The term "isolated nucleic acid" includes, for example, a recombinant DNA which is incorporated into a vector, such as a plasmid or virus; or incorporated into the genomic DNA of a prokaryoie or eukaryote; or which exists as a separate molecule. [0048] A synthetic CRISPR/cas locus is expressed in a cell by methods well known in the art. As used herein, the term "express," "expressed," or "expressing" a gene or protein refers to the introduction of a nucleic acid or protein, or the presence of a nucleic acid or protein, in a ceil. Expression may be at the nucleic acid or protein level. In some embodiments, the cell is transfected with the nucleic acid.

[0049] As used herein, the term "modulate" refers to a. change or an alteration in expression or activity of a gene. Modulation may be an increase or a decrease in expression. In some embodiments, modulation means that expression or activity of a gene is diminished, reduced, inhibited, or silenced. In other embodiments, modulation means that the expression of a gene is augmented, for example, turned on or increased. In some embodiments, the expression of the gene is reduced or increased by about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more. Expression of one or more genes is measured by methods well known in the art that include, but are not limited to, Northern blot, PGR, in situ hybridization, RNA-seq, and dot-blotting. In some embodiments, expression is reduced or increased by at least 1 % as measured by Northern blot. In some embodiments, expression is reduced or increased by at least 10% as measured by Northern blot. In other embodiments, expression is reduced or increased by at least 50% as measured by Northern blot.

[0050] The phrases "off-target effects" and "non-specific effects" are used interchangeably herein and refer to undesirable effects of a composition on genes and proteins that the composition is not designed to target. In one embodiment, the compositions described herein demonstrate reduced off target effects as compared to gene silencing techniques commonly known in the art. Off-target effects can be measured via methods known by those skilled in the art, including microarray analysis or RNA-seq (i.e., whole-transcriptome shotgun sequencing; see, for example, Qi et ah, 2013 Cell 152; 1 173-83). To measure the number of differentially expressed genes. The number of differentially expressed genes may be expressed as the frequency of off-target effects. A differentially expressed gene may be defined as a gene having at least a 2-fold change in expression after treatment. The change in expression may be an increase or a decrease in expression. [0051] The term "eukaryote" as used herein refers to an organism with a nucleus and complex cellular structures enclosed within membranes. In some embodiments, the eukaryote is a yeast, metazoan, or animal. In further embodiments, the animal is a mammal. In further embodiments, the mammal may be human, non-human primate, canine, feline, rodent, bovine, equine, porcine, or any other mammal. The term "prokaryote" as used herein refers to organisms such as bacteria and archaea that have cells lacking a nucleus.

[0052] A "vector" is a composition of matter, which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasrnids, and viruses. Thus, the term "vector" includes an autonomously replicating or non-replicating plasmid or a virus. Examples of viral vectors include, but are not, limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. An expression construct can be replicated in a living cell, or it can be made synthetically. For purposes of this application, the terms "expression construct," "expression vector," and "vector," are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.

[0053] As used herein, a "promoter" refers to a DNA sequence recognized by the machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. Suitable promoters include, but are not limited to RNA pol I, pol II, pol III, and viral promoters (e.g. human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In one embodiment, the promoter is a tissue specific promoter. The promoter together with other transcriptional and translational regulatory nucleic acid sequences is necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. The term "promoter" also refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. [0054] In one embodiment, a vector comprises a promoter that is operably linked to a polynucleotide. The phrase "operably linked" as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide. Thus, in some embodiments, the polynucleotide is under transcriptional control of a promoter. In certain embodiments, the promoter operably linked to a polynucleotide may be an inducible promoter. Inducible promoters are known in the art and include, but, are not limited to, tetracycline promoter, metallothionein IIA promoter, heat, shock promoter, steroid/thyroid hormone/retinoic acid response elements, the adenovirus late promoter, and the inducible mouse mammary tumor virus LTR.

[0055] In one embodiment, the present invention provides a method of modulating the expression of one or more target DNA sequences comprising expressing a synthetic CRISPR cas system comprising a CR ISPR locus comprising spacer sequences complementary to the one or more target DNA sequences, wherein said spacer sequences hybridize to said one or more target DNA sequences, and wherein expression of the one or more target DNA sequences is turned on or increased. In one embodiment, the CRISPR/cas system comprises one or more protein that is tethered to a transcriptional initiation factor. "Tethered," as used herein, refers attachment or connection of two compositions (e.g., a CRISPR-related protein and a transcriptional initiation factor) and may be used interchangeably herein with "fused" or "linked." Transcriptional initiation factors for prokaryotic transcription are well known in the art and include sigma factors

(e.g., σ 19 , σ 2 \ σ , σ 3i , σ j8 , and σ * ). Transcriptional initiation factors for eiikaryotic transcription are well known in the art and include, for example, STAT family proteins (e.g., STATs 1 , 2, 3, 4, 5, and 6), fos/jun, NFkappaB, HIV-Tat, and the E2F family. In one embodiment, the transcriptional initiation factor is tethered to the C-terminus or the N-terminus of the CRISPR protein. In one embodiment, the transcriptional initiation factor is tethered to CasA, CasB, CasC, CasD, or CasE. In certain embodiments, the transcriptional initiation factor is tethered to CasA or CasE. In further embodiments, the transcriptional initiation factor is tethered to the C-terminus of CasA or the N-terminus of CasE. In certain embodiments, the transcriptional activation factor and is tethered in such a way that will permit DNA binding and RNA polymerase recruitment without steric clashing. Thus, in one embodiment, the CRISPR/cas locus directs expression of the target sequence,

[0056] In one embodiment, the synthetic CRISPR comprises a nuclear localization sequence. In a further embodiment, the nuclear localization sequence directs the CRISPR to the nucleus of a eukaryotic cell. As used herein, the term "nuclear localization signal" is used interchangeably with the term "nuclear localization sequence" and refers to an amino acid sequence, which directs a target protein to the cell nucleus by nuclear transport. In some embodiments, the signal comprises one or more short, amino acid sequences of positively charged lysines or arginines. Exemplary nuclear localization signals include, but are not limited to, those from the SV40 Large T antigen or SV40 medium T-antigen, influenza virus, and viral Tat proteins such as HIV Tat.

[0057] In some embodiments, the synthetic CRISPR. targets a single site in a genome or a single gene within a cell. In other embodiments, the synthetic CRISPR or CRISPRs are capable of targeting multiple sites in a genome or multiple genes within a cell. In other embodiments, the CRISPR or CRISPRs are capable of targeting multiple sites within a single gene. In some embodiments, a CR ISPR is designed to target one or more promoters. Without wishing to be bound by theory, CRISPRs designated to target promoters will block promoter recognition by transcriptional initiators, and these sites will be the most potent inhibitors.

[00S8| "Complementary" is used herein to refer to the extent that a polynucleotide sequence is identical to the reverse-complement of ail or a portion of a reference polynucleotide sequence. Thus, a sequence is 100% complementary to a reference sequence if 100% of the nucleotides of the first sequence are able to form a base-pair with nucleotides of the reference sequence; and a sequence is 90% complementary to a reference sequence if 9 out of 10 nucleotides are able to form a base-pair with the nucleotides of the reference sequence. For illustration, the nucleotide sequence "5'-TATAC-3"' is 100% complementary to a reference sequence "5'-GTATA-3"'. For further illustration, the nucleotide sequence "5'-TATAC-3"' is 80% complementary to a reference sequence "5'-GTATG-3"\ In some embodiments, the synthetic CRISPR/cas loci comprise sequences that are 100% complementary to their DNA targets. In other embodiments, the synthetic CRISPR/cas loci comprise sequences that are not 100% complementary to their DNA targets. For example, in some embodiments, the synthetic CRISPR/cas loci comprise sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%), 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to their DNA targets. In some embodiments, transcriptional repression by CRISPR/cas loci is tunable based on the use of CRISPRs that are not 100% complementary to their DNA targets. For example, transcriptional repression or activation in some embodiments is decreased when CRISPR/cas loci contain sequences that are less than 100% complementary to the target DNA sequence. In some embodiments, transcriptional repression or activation by CRISPR/cas loci is tunable based on the number of target sites in a single gene. For example, repression or activation by CRISPR of a particular gene is increased when more than one site on the gene is targeted. In one embodiment, a CRISPR. may comprise spacer sequences complementary to 1 , 2, 3, 4, 5, 6, 7, 8 or more sites on a gene.

[0059] In one embodiment, the CRISPR/cas systems useful in the invention are Cascade or Cascade-like CRISPR/cas complexes. Cascade-like CRISPR/cas systems include any CRISPR-associated ribonucleoprotein including, but not limited to, Cascade, aCASCADE, the Csy-complex, and 1-C/Dvulg Cascade. The terms "CRISPR," "CRISPRs," "CRISPR/cas locus," and "CR ISPR/cas loci," are used interchangeably herein and refer to a clustered regularly interspaced short palindromic repeat. The terms "CRISPR/cas system" and CRISPR/cas complex" are used interchangeably herein and refer to nucleic-acid-based adaptive immune system found in or derived from bacteria or archaea. Each CRISPR/cas system or complex is comprised of the CRISPR/cas locus and one or more Cas preoteins encoded by a cassette of CRISPR-associated (cas) genes. The terms "spacer sequence" and "crRNAs" ("CRISPR-derived RNAs") are used interchangeably herein and refer to the library of short RNAs that contain unique sequences complementary to a foreign nucleic acid. "Synthetic CRISPR" or "synthetic CRISPR/cas loci" or "synthetic CRISPR/cas system" refer to CRISPRs that have been engineered to target a particular sequence. Synthetic CRISPRs may be engineered such that they do not include all Cas proteins associated with the CRISPR Type. Synthetic CRISPRs may further comprise transcription factors or transcription initiation factors, nuclear localization factors, or other factors involved in the transcription of sequences. Synthetic CRISPRs may be engineered such that different components of the CRISPR/cas locus are present on different expression vectors. For example, the cas genes, or cas gene cassette, may be present on a separate expression vector comprising the DNA targeting sequences. I one embodiment, the CR1SPR complex self-assembles upon expression of components of the CRISPR/cas locus and the cas genes in a host cell.

[0060] The terms "treat," "treatment," and "treating" refer to an approach for obtaining beneficial or desired results, for example, clinical results. For the purposes of this invention, beneficial or desired results may include inhibiting or suppressing the growth of an infectious agent or killing an infectious agent; inhibiting one or more processes through which an infectious agent infects a cell or subject; inhibiting or ameliorating the disease or condition caused by an infectious agent; inhibiting or ameliorating the symptoms of a disease caused by an infectious agent; or a combination thereof. Beneficial or desired results may also include inhibiting or suppressing the development or progression of a disease or disorder that is caused in whole or in part by a target DNA sequence. In some embodiments, the target DNA sequence is associated with an infectious agent. In other embodiments, the target DNA sequence is not associated with an infectious agent. In embodiments, the subject, plant, or animal has a disease or condition caused in whole or in part by said target DNA sequence. By "has a disease or condition caused in whole or in part by the target DNA sequence," is meant that inappropriate, excessive, or deficient expression of the gene is frequently, typically, or consistently occurs in the presence of the disease or condition. The terms "treat," "treatment," or "treating" also refer to prophylaxis or prevention of an infection, disease, or disorder, as well as ameliorating of the symptoms of the infection, disease, or disorder. The term "subject," as used herein, may refer to animals, yeasts, metazoa, bacteria, archaea, cell lines such as human cell lines (e.g., HeLa, 293T ceils, RAW), embryonic stem cells, or hamster cells (e.g., CHO). As used herein, the term "animal" refers to humans, non-human primates, sheep, goats, cattle, pigs, deer, elk, clogs, buffaloes, camels, horses, mules, donkeys, cats, bison, both wild-life and domestic, bison/cattle hybrids (beefalo and/or cattalo), antelope, bears, rodents (including mice and rats), monkeys, rabbits, reindeer, caribou, fish, birds, chickens, roosters, ducks, geese, turkeys, oxen, Llamas, alpacas, emus, ostriches, honey bees and other insects.

[0061] The use of the word "a" or "an" when used in conjunction with the term

"comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention. Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[0062] As used in this specification and ciaim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form, of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form, of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0063] This invention is further illustrated by the following additional examples that should not be construed as limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples

Example 1. Expression of Cascade complex

[0064] The five cas genes were cloned into three different expression vectors and synthetic CRISPR/cas locus repeats containing seven identical spacer sequences were cloned downstream of a T7 promoter on a fourth vector that does not contain a ribosome binding site

(Table 1). The CasB subunit was engineered to include an N-terminal Strep II tag followed by a

Precision protease cleavage site. These four expression vectors were co-transformed into BL21

DE3 E. coli ceils, cultured in selective media and overexpression of the complex was induced with 0.5 mM IPTG. The self-assembled Cascade complex was purified using a simple four step process involving Step-Tactin affinity purification, Prescission protease cleavage of the Strep II tag, a second Step-Taetin pulldown for removal of liberated Strep II tag and size exclusion chromatography. In our experience we recover ~15 mgs of the purified complex per liter of E. coli culture (Figure 1). The complex is active (binds DNA targets with low nanomolar affinities) and the yields are more than sufficient for biochemical and structural studies.

Table 1. Expression constructs for Cascade

Example 2. Alternatives to iiolo Cascade complex from E. coli

[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 3z P-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 σ 32 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 <f 2 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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