CRISPR-CAS SYSTEM MATERIALS AND METHODS

• US 20160298096A1
• Filed: 11/17/2014
• Published: 10/13/2016
• Est. Priority Date: 11/18/2013
• Status: Abandoned Application

• Current Assignee: CRISPR Therapeutics AG
• Original Assignee: CRISPR Therapeutics AG
• Inventors: Fontara, Ines, Charpentier, Emmanuelle, Chylinski, Krzysztof
• Application Number:
  US15/037,371
• Publication Number:
  US 20160298096A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

The invention relates to Type II CRIS-PR-Cas systems of Cas9 enzymes, guide RNAs and associated specific PAMs.

Claims

1. A single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment wherein the protein-binding segment comprises a tracrRNA set out in Supplementary Table S5.

2. The single-molecule guide RNA of claim 1 wherein the protein-binding segment comprises a CRISPR repeat set out in Supplementary Table S5 that is the cognate CRISPR repeat of the tracrRNA of the protein-binding segment.

3. The single-molecule guide RNA of claim 1 or 2 wherein the DNA-targeting segment further comprises RNA complementary to a protospacer-like sequence in a target DNA 5′

to a PAM sequence.

4. The single-molecule guide RNA of claim 3 wherein the tracrRNA and CRISPR repeat are respectively at least 80% identical to the C. jejuni tracrRNA and CRISPR repeat set out in Supplementary Table S5 and wherein the PAM sequence is NNNNACA.

5. The single-molecule guide RNA of claim 4 or 8 wherein the RNA complementary to a protospacer-like sequence is RNA complementary to the target sequences set out in one of SEQ ID NOs:

801-973, 1079-1222, 1313-1348, 1372-1415, 1444-1900, 2163-2482 or 2667-2686.

9. The single-molecule guide RNA of claim 1 or 6 comprising a linker between the DNA-targeting segment and the protein-binding segment.

6. A single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5.

7. A single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5, a CRISPR repeat at least 80% identical to a CRISPR repeat set out in Supplementary Table S5, or both.

8. The single-molecule guide RNA of claim 7 wherein the tracrRNA and CRISPR repeat are respectively at least 80% identical to the C. jejuni tracrRNA and CRISPR repeat set out in Supplementary Table S5 and wherein the PAM sequence is NNNNACA.

10. A DNA encoding a single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA set out in Supplementary Table S5.

11. A DNA encoding a single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5, a CRISPR repeat at least 80% identical to a CRISPR repeat set out in Supplementary Table S5, or both.

12. A vector comprising a DNA encoding a single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment wherein the protein-binding segment comprises a tracrRNA set out in Supplementary Table S5.

13. A vector comprising a DNA encoding a single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5, a CRISPR repeat at least 80% identical to a CRISPR repeat set out in Supplementary Table S5, or both.

14. A cell comprising a DNA encoding a single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA set out in Supplementary Table S5.

15. A cell comprising a DNA encoding a single-molecule guide RNA comprising:

a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5, a CRISPR repeat at least 80% identical to a CRISPR repeat set out in Supplementary Table S5, or both.

16. A double-molecule guide RNA comprising:

a targeter-RNA and an activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA set out in Supplementary Table S5, and

wherein the guide RNA comprises a modified backbone, a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, a base modification, a modification or sequence that provides for modified or regulated stability, a modification or sequence that provides for subcellular tracking, a modification or sequence that provides for tracking, or a modification or sequence that provides for a binding site for a protein or protein complex.

17. The double-molecule guide RNA of claim 16, wherein the targeter-RNA comprises a CRISPR repeat set out in Supplementary Table S5 that is the cognate CRISPR repeat of the tracrRNA of the protein-binding segment.

18. The double-molecule guide RNA of claim 16 or 17 wherein the targeter-RNA further comprises RNA complementary to a protospacer-like sequence in a target DNA 5′

to a PAM sequence.

19. The double-molecule guide RNA of claim 18 wherein the tracrRNA and CRISPR repeat are respectively at least 80% identical to the C. jejuni tracrRNA and CRISPR repeat set out in Supplementary Table S5 and wherein the PAM sequence is NNNNACA.

20. The double-molecule guide RNA of claim 19 or claim 23 wherein the RNA complementary to a protospacer-like sequence is RNA complementary to the target sequences set out in one of SEQ ID NOs:

801-973, 1079-1222, 1313-1348, 1372-1415, 1444-1900, 2163-2482 or 2667-2686.

24. The double-molecule guide RNA of claim 16 or 21 comprising a linker between the targeter-RNA and the activator-RNA.

21. A double-molecule guide RNA comprising:

a targeter-RNA and a activator-RNA, wherein the activator-RNA comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5.

22. The double-molecule guide RNA of claim 21, wherein the targeter-RNA comprises a CRISPR repeat set out in Supplementary Table S5, the cognate CRISPR repeat of the tracrRNA of the activator-RNA set out in Supplementary Table S5, or a CRISPR repeat at least 80% identical to a CRISPR repeat set out in Supplementary Table S5.

23. The double-molecule guide RNA of claim 21 wherein the tracrRNA and CRISPR repeat are respectively at least 80% identical to the C. jejuni tracrRNA and CRISPR repeat set out in Supplementary Table S5 and wherein the PAM sequence is NNNNACA.

25. A DNA encoding a double-molecule guide RNA comprising:

a targeter-RNA and a activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA set out in Supplementary Table S5.

26. A DNA encoding a double-molecule guide RNA comprising:

a targeter-RNA and a activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5, a CRISPR repeat at least 80% identical to a CRISPR repeat set out in Supplementary Table S5, or both.

27. A vector comprising a DNA encoding a double-molecule guide RNA comprising:

a targeter-RNA and a activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA set out in Supplementary Table S5.

28. A vector comprising a DNA encoding a double-molecule guide RNA comprising:

a targeter-RNA and a activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5, a CRISPR repeat at least 80% identical to a CRISPR repeat set out in Supplementary Table S5, or both.

29. A cell comprising a DNA encoding a double-molecule guide RNA comprising:

a targeter-RNA and a activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA set out in Supplementary Table S5.

30. A cell comprising a DNA encoding a double-molecule guide RNA comprising:

a targeter-RNA and a activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5, a CRISPR repeat at least 80% identical to a CRISPR repeat set out in Supplementary Table S5, or both.

31. A method for manipulating DNA in a cell, comprising contacting the DNA with a Cas9 ortholog-guideRNA complex, wherein the complex comprises:

(a) a C. jejuni Cas9 endonuclease heterologous to the cell or an endonuclease with an activity portion at least 90% identical to the activity portion of the C. jejuni Cas9 endonuclease, and a guide RNA targeting the complex to a protospacer-like sequence in the DNA 5′

to the PAM sequence NNNNACA;

(b) a P. multocida Cas9 endonuclease heterologous to the cell or an endonuclease with an activity portion at least 90% identical to the activity portion of the P. multocida Cas9 endonuclease, and a guide RNA targeting the complex to a protospacer-like sequence in the DNA 5′

to the PAM sequence GNNNCNNA or NNNNC;

(c) an F. novicida Cas9 endonuclease heterologous to the cell or an endonuclease with an activity portion at least 90% identical to the activity portion of the F. novicida Cas9 endonuclease, and a guide RNA targeting the complex to a protospacer-like sequence in the DNA 5′

to the PAM sequence NG;

(d) an S. thermophilus** Cas9 endonuclease heterologous to the cell or an endonuclease with an activity portion at least 90% identical to the activity portion of the S. thermophilus** Cas9 endonuclease, and a guide RNA targeting the complex to a protospacer-like sequence in the DNA 5′

to the PAM sequence NNAAAAW;

(e) an L. innocua Cas9 endonuclease heterologous to the cell or an endonuclease with an activity portion at least 90% identical to the activity portion of the L. innocua Cas9 endonuclease, and a guide RNA targeting the complex to a protospacer-like sequence in the DNA 5′

to the PAM sequence NGG;

or

(f) an S. dysgalactiae Cas9 endonuclease heterologous to the cell or an endonuclease with an activity portion at least 90% identical to the activity portion of the S. dysgalactiae Cas9 endonuclease, and a guide RNA targeting the complex to a protospacer-like sequence in the DNA 5′

to the PAM sequence NGG.

32. The method of claim 31 wherein the cell is a bacterial cell, a fungal cell, an archaea cell, a plant cell or an animal cell.

33. The method of claim 31 wherein the guide RNA is a single-molecule guide RNA.

34. The method of claim 31 wherein the guide RNA is a double-molecule guide RNA.

35. The method of claim 31 wherein the endonuclease is a nickase.

36. The method of claim 31 wherein the endonuclease comprises a mutation corresponding to S pyogenes E762A, HH983AA or D986A.

37. The method of claim 31 wherein the endonuclease is a dead mutant/DNA binding protein.

38. The method of claim 31 wherein the protospacer-like sequence targeted is in a CCR5, CXCR4, KRT5, KRT14, PLEC or COL7A1 gene.

39. The method of claim 31 wherein the protospacer-like sequence is in a chronic granulomatous disease (CGD)-related gene CYBA, CYBB, NCF1, NCF2 or NCF4.

40. The method of claim 31 wherein the protospacer-like sequence targeted is in, or is up to 1000 nucleotides upstream of, a gene encoding B-cell lymphoma/leukemia IIA (BCL11A) protein, an erythroid enhancer of BCL11A or a BCL11A binding site.

41. The method of claim 31 wherein the endonuclease and the guide RNA are introduced to the cell by the same or different recombinant vectors encoding the endonuclease and the guide RNA.

42. The method of claim 31 wherein at least one recombinant vector is a recombinant viral vector.

43. A recombinant vector encoding:

(a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′

to the PAM sequence NNNNACA; and

(b) s C. jejuni Cas9 endonuclease or an endonuclease with an activity portion at least 90% identical to the activity portion of the C. jejuni Cas9 endonuclease.

49. The recombinant vector of claim 43, 44, 45, 46, 47 or 48 wherein the recombinant vector is a recombinant viral vector.

44. A recombinant vector encoding:

(a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′

to the PAM sequence GNNNCNNA or NNNNC; and

(b) a P. multocida Cas9 endonuclease or an endonuclease with an activity portion at least 90% identical to the activity portion the P. multocida Cas9 endonuclease.

45. A recombinant vector encoding:

(a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′

to the PAM sequence NG; and

(b) a F. novicida Cas9 endonuclease or an endonuclease with an activity portion at least 90% identical to the activity portion of the F. novicida Cas9 endonuclease.

46. A recombinant vector encoding:

(a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′

to the PAM sequence NNAAAAW; and

(b) a S. thermophilus** Cas9 endonuclease or an endonuclease with an activity portion at least 90% identical to the activity portion of the S. thermophilus** Cas9 endonuclease.

47. A recombinant vector encoding:

(a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′

to the PAM sequence NGG; and

(b) a L. innocua Cas9 endonuclease or an endonuclease with an activity portion at least 90% identical to the activity portion of the L. innocua Cas9 endonuclease.

48. A recombinant vector encoding:

(a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′

to the PAM sequence NGG; and

(b) a S. dysgalactiae Cas9 endonuclease or an endonuclease with an activity portion at least 90% identical to the activity portion of the S. dysgalactiae Cas9 endonuclease.

50. A modified Cas9 endonuclease comprising one or more mutations corresponding to S. pyogenes mutation E762A, HH983AA or D986A.

51. The modified Cas 9 endonuclease of claim 50 further comprising one or more mutations corresponding to S. pyogenes mutation D10A, H840A, G12A, G17A, N854A, N863A, N982A or A984A.

52. A method for manipulating DNA in a cell, comprising contacting the DNA with a Cas9 ortholog-guide RNA complex, wherein the complex comprises:

(a) a Cas9 endonuclease heterologous to the cell and

(b) a cognate guide RNA of the Cas9 endonuclease comprising a tracrRNA set out in Supplementary Table S5 or a guide RNA comprising a tracrRNA at least 80% identical to a cognate tracrRNA set out in Supplementary Table S5 over at least 20 nucleotides.

53. The method of claim 52 wherein the cell is a bacterial cell, a fungal cell, an archaea cell, a plant cell or an animal cell.

54. The method of claim 52 wherein the guide RNA is a single-molecule guide RNA.

55. The method of claim 52 wherein the guide RNA is a double-molecule guide RNA.

56. The method of claim 52 wherein the endonuclease is a nickase.

57. The method of claim 52 wherein the endonuclease comprises a mutation corresponding to S pyogenes mutations E762, HH983AA or D986A.

58. The method of claim 52 wherein the endonuclease is a dead mutant/DNA binding protein.

59. The method of claim 52 wherein the protospacer-like sequence targeted is in a CCR5, CXCR4, KRT5, KRT14, PLEC or COL7A1 gene or a sequence up to 1000 nucleotides upstream of the gene.

60. The method of claim 52 wherein the protospacer-like sequence is in a chronic granulomatous disease (CGD)-related gene CYBA, CYBB, NCF1, NCF2 or NCF4 or a sequence up to 1000 nucleotides upstream of the gene.

61. The method of claim 52 wherein the protospacer-like sequence targeted is in, or is up to 1000 nucleotides upstream of, a gene encoding B-cell lymphoma/leukemia IIA (BCL11A) protein, an erythroid enhancer of BCL11A or a BCL11A binding site.

62. The method of claim 52 wherein the endonuclease and the guide RNA are introduced to the cell by the same or different recombinant vectors encoding the endonuclease and the guide RNA.

63. The method of claim 52 wherein at least one recombinant vector is a recombinant viral vector.