CRISPR enzymes and systems

• US 9,790,490 B2
• Filed: 12/18/2015
• Issued: 10/17/2017
• Est. Priority Date: 06/18/2015
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Zhang, Feng, Zetsche, Bernd, Slaymaker, Ian, Gootenberg, Jonathan, Abudayyeh, Omar O.
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US14/975,085
• Publication Number:
  US 20160208243A1
• Time in Patent Office: 669 Days
• Field of Search: None
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/82 for plant cells , e.g. plant artificial chromosomes (PACs)
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/16 Aptamers
• 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 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

Claims

1. An engineered, non-naturally occurring system comprising

a) a Cpf1 effector protein, and

b) at least one engineered guide polynucleotide designed to form a complex with the Cpf1effector protein and comprising a guide sequence, wherein the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell;

and

wherein the system lacks a tracr sequence, the engineered guide polynucleotide and Cpf1 effector protein do not naturally occur together, and a complex of the engineered guide polynucleotide and Cpf1 effector protein does not naturally occur.

9. The system according to claim 1, 2, 3, or 4, wherein the complex of the at least one engineered guide polynucleotide and the Cpf1 effector protein causes cleavage distally of the target sequence.

10. The system according to claim 9, wherein said cleavage generates a staggered double stranded break with a 4- or 5-nt 5′

overhang.

11. The system according to claim 1, 2, 3, or 4, wherein said target sequence is 3′

of a Protospacer Adjacent Motif (PAM) and the PAM comprises a 5′

T-rich motif.

12. The system according to claim 1, 2, 3, or 4, wherein the Cpf1 effector protein is of a bacterial species listed in FIG. 64A or FIG. 64B.

13. The system according to claim 12, wherein the bacterial species is selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae.

14. The system according to claim 1, 2, 3, or 4, wherein said target sequence is 3′

of a Protospacer Adjacent Motif (PAM) and the PAM sequence is TTN, where N is A/C/G or T and the Cpf1 effector protein is FnCpf1, or the PAM sequence is TTTV, where V is A/C or G and the Cpf1 effector protein is PaCpf1p, LbCpf1 or AsCpf1.

15. The system according to claim 1, 2, 3, or 4, wherein the Cpf1 effector protein comprises one or more nuclear localization signals.

19. The system according to claim 1, 2, 3, or 4, wherein the eukaryotic cell is an animal or human cell.

20. The system according to claim 1, 2, 3, or 4, wherein the eukaryotic cell is a plant cell.

21. The system according to claim 1, 2, 3, or 4, wherein the guide sequence is linked to a direct repeat sequence.

22. The system according to claim 1, 2, 3, or 4, wherein the complex, engineered guide polynucleotide or Cpf1 effector protein is conjugated to at least one sugar moiety.

23. The system according to claim 22, wherein the sugar moiety comprises N-acetyl galactosamine (GalNAc) or triantennary GalNAc.

24. The system according to claim 1, 2, 3, or 4, wherein the engineered guide polynucleotide comprises at least one modified nucleotide.

25. A delivery particle comprising the system according to claim 1, 2, 3, or 4.

26. The delivery particle of claim 25, wherein the Cpf1 effector protein is complexed with the engineered guide polynucleotide.

27. The delivery particle of claim 25, further comprising a lipid, a sugar, a metal or a protein.

28. The delivery particle of claim 26, further comprising a lipid, a sugar, a metal or a protein.

29. A method of modifying a locus of interest having a target sequence of a eukaryotic cell comprising delivering the system according to claim 1, 2, 3, or 4, to said locus of interest, wherein the guide sequence hybridizes with the target sequence whereby modification of the locus of interest occurs.

30. The method of claim 29, wherein the locus of interest is within a eukaryotic cell.

31. The method of claim 30, wherein the cell is an animal cell.

32. The method of claim 30, wherein the cell is an animal or human cell.

33. The method of claim 30, wherein the cell is a plant cell.

34. The method of claim 30, wherein said delivering the system comprises delivering one or more nucleotide sequences encoding the Cpf1 effector protein and one or more nucleotide sequences encoding the at least one engineered guide polynucleotide.

35. The method of claim 34, wherein the one or more nucleotide sequences are comprised within one or more vectors.

36. The method of claim 34, wherein the one or more nucleotide sequences are comprised within a single vector.

37. The method of claim 34, wherein the one or more nucleotide sequences molecules are delivered via delivery particles, vesicles, or one or more viral vectors.

38. The method of claim 30, wherein the system or a component thereof is delivered via delivery particles, delivery vesicles, or one or more viral vectors.

39. The method of claim 38, wherein the system is delivered via delivery particles, delivery vesicles, or one or more viral vectors.

40. The method of claim 38, wherein the delivery particles comprise a lipid, a sugar, a metal or a protein.

41. The method of claim 38, wherein the delivery vesicles are exosomes or liposomes.

42. The method of claim 38, wherein the one or more viral vectors are one or more adenoviruses, one or more lentiviruses or one or more adeno-associated viruses.

43. The method of claim 30, wherein the Cpf1 effector protein comprises one or more nuclear localization signals.

44. The method of claim 30, wherein the eukaryotic cell comprises a cell of a cell line or a cell of an organism and the locus of interest comprises a genomic locus of interest.

45. The method of claim 30, wherein the eukaryotic cell is in vitro, ex vivo or in vivo.

46. The method of claim 30, wherein the eukaryotic cell comprises a stem cell or stem cell line.

47. The method of claim 29, wherein the locus of interest is within a eukaryotic DNA molecule in vitro.

48. The method of claim 33, wherein the method comprises obtaining a plant having a modified trait of interest from the plant cell.

49. The method of claim 33, wherein the method comprises identifying a trait of interest in a plant obtained from the plant cell, wherein the trait of interest is encoded by a gene of interest.

50. The method of claim 49, further comprising introducing the identified gene of interest into a plant cell or a plant cell line or plant germplasm and generating therefrom a plant containing the gene of interest.

51. The method of claim 50, wherein the plant exhibits the trait of interest.

52. The method of claim 29, wherein the modification comprises a strand break.

53. The method of claim 52, wherein the strand break comprises a staggered DNA double stranded break with a 4- or 5-nt 5′

overhang.

54. The method of claim 53, wherein the modification comprises integration of a DNA insert into the staggered DNA double stranded break.

55. The method of claim 29, including adding a cation.

56. A eukaryotic cell comprising the system of any one of claims 1-8.

57. The cell of claim 56, wherein the eukaryotic cell is an animal cell.

58. The cell of claim 56, wherein the eukaryotic cell is a human cell.

59. The cell of claim 56, wherein the eukaryotic cell comprises a stem cell or stem cell line.

60. The cell of claim 56, wherein the eukaryotic cell is a plant cell.

2. An engineered, non-naturally occurring system comprising

a) one or more nucleotide sequences encoding a Cpf1 effector protein, and

b) one or more nucleotide sequences encoding at least one engineered guide polynucleotide designed to form a complex with the Cpf1 effector protein and comprising a guide sequence, wherein the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell;

and

wherein the system lacks a tracr sequence, the engineered guide polynucleotide and Cpf1 effector protein do not naturally occur together, and a complex of the engineered guide polynucleotide and Cpf1 effector protein does not naturally occur.

16. The system according to claim 2, 3, or 4, wherein the one or more nucleotide sequences encoding the Cpf1 effector protein is/are codon optimized for expression in a eukaryotic cell.

17. The system according to claim 2, 3, or 4, wherein components (a) and (b) are on one vector.

18. The system according to claim 2 or 4, wherein the one or more nucleotide sequences of components (a) and (b) are a single nucleic acid molecule.

3. An engineered, non-naturally occurring vector system comprising one or more vectors comprising

a) a first regulatory element operably linked to one or more nucleotide sequences encoding a Cpf1 effector protein;

b) a second regulatory element operably linked to one or more nucleotide sequences encoding at least one engineered guide polynucleotide designed to form a complex with the Cpf1 effector protein and comprising a guide sequence, wherein the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell; and

wherein the system lacks a tracr sequence, components (a) and (b) are located on the same or different vectors of the system, the engineered guide polynucleotide and Cpf1 effector protein do not naturally occur together, and a complex of the engineered guide polynucleotide and Cpf1 effector protein does not naturally occur.

4. An engineered, non-naturally occurring system comprising

a) a Cpf1 effector protein, or one or more nucleotide sequences encoding the Cpf1 effector protein, and

b) at least one engineered guide polynucleotide designed to form a complex with the Cpf1 effector protein and comprising a guide sequence, wherein the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell, or one or more nucleotide sequences encoding the at least one engineered guide polynucleotide;

wherein the system lacks a tracr sequence, the engineered guide polynucleotide and Cpf1 effector protein do not naturally occur together, and a complex of the engineered guide polynucleotide and Cpf1 effector protein does not naturally occur.

5. The system according to claim 4, comprising the at least one engineered guide polynucleotide.

6. The system according to claim 4, comprising the one or more nucleotide sequences encoding the Cpf1 effector protein.

7. The system according to claim 4, comprising the Cpf1 effector protein.

8. The system according to claim 4, comprising the one or more nucleotide sequences encoding the at least one engineered guide polynucleotide.