Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

• US 10,266,850 B2
• Filed: 03/15/2013
• Issued: 04/23/2019
• Est. Priority Date: 05/25/2012
• Status: Active Grant

• Current Assignee: The University Of Vienna, Regents of the University of California (University of California)
• Original Assignee: Emmanuelle Charpentier, The University Of Vienna, Regents of the University of California (University of California)
• Inventors: Doudna, Jennifer A., Jinek, Martin, Charpentier, Emmanuelle, Chylinski, Krzysztof
• Primary Examiner(s): Hammell, Neil P
• Application Number:
  US13/842,859
• Publication Number:
  US 20140068797A1
• Time in Patent Office: 2,230 Days
• Field of Search: None
• CPC Class Codes: A01H 6/4684 Zea mays [maize]
• CPC Class Codes: A01K 67/027 New or modified breeds of vertebrates
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• 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 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/20 : involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/31 : of the backbone
• CPC Class Codes: C12N 2310/32 : of the sugar
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/3519 : Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/531 : Stem-loop; Hairpin
• CPC Class Codes: C12N 2800/80 : Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Y 301/04 : Phosphoric diester hydrolases (3.1.4)
• CPC Class Codes: H01L 21/02312 : treatment by exposure to a gas or vapour
• CPC Class Codes: H01L 21/02365 : Forming inorganic semiconducting materials on a substrate for light-sensitive devices H01L31/00
• CPC Class Codes: H01L 33/0075 : comprising nitride compounds
• 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 present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Claims

1. A method of cleaving a nucleic acid comprising

contacting a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising

a) a Cas9 protein;

and

b) a single molecule DNA-targeting RNA comprising

i) a targeter-RNA that hybridizes with the target sequence, and

ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,

wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides,

wherein the single molecule DNA-targeting RNA forms a complex with the Cas9 protein,

whereby the single molecule DNA-targeting RNA targets the target sequence, and the Cas9 protein cleaves the target DNA molecule.

2. An engineered and/or non-naturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising

a Cas9 protein or a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, and

a single molecule DNA-targeting RNA or a nucleic acid comprising a nucleotide sequence encoding the single molecule DNA-targeting RNA;

wherein the single molecule DNA-targeting RNA comprises

i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and

ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,

wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides, and

wherein the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein,

whereby hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule.

3. The system of claim 2, wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein and/or the nucleic acid comprising the nucleotide sequence encoding the single molecule DNA-targeting RNA are one or more vectors.

4. A method of cleaving or editing a target DNA molecule or modulating transcription of at least one gene encoded thereon, the method comprising

contacting a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising:

a) a single molecule DNA-targeting RNA comprising

i) a targeter-RNA that hybridizes with the target sequence, and

ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,

wherein the targeter-RNA and the activator-RNA are covalently linked to one another with intervening nucleotides; and

b) a Cas9 protein,

wherein the single molecule DNA-targeting RNA forms a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule,

whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated.

5. The method of claim 4, wherein the single molecule DNA-targeting RNA is transcribed from a vector comprising a nucleotide sequence encoding the single molecule DNA-targeting RNA.

6. The method of claim 5, wherein the nucleotide sequence is operably linked to a control element operable in a desired cell type.

7. The method of claim 5, wherein the vector is selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors.

8. The method of claim 7, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors.

9. The method of claim 4, wherein the Cas9 protein is transcribed and translated from a vector comprising a nucleotide sequence encoding the Cas9 protein.

10. The method of claim 9, wherein the nucleotide sequence is operably linked to a control element operable in a desired cell type.

11. The method of claim 9, wherein the vector is selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors.

12. The method of claim 11, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors.

13. The method of claim 4, wherein the CRISPR-Cas system comprises two or more single molecule DNA-targeting RNAs.

14. The method of claim 4, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a non-homologous end joining (NHEJ) repair mechanism, thereby editing the target DNA molecule.

15. The method of claim 4, wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the target DNA molecule, thereby editing the target DNA molecule.

16. The method of claim 4, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).

17. The method of claim 16, wherein the one or more PTD(s) aid in traversal of an organelle membrane.

18. The method of claim 16, wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

268 and 269.

19. The method of claim 4, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.

20. The method of claim 4, wherein the Cas9 protein is transcribed and translated from a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, wherein the nucleotide sequence is modified relative to a corresponding wild-type nucleotide sequence, wherein the modification replaces one or more codons in the wild-type nucleotide sequence with one or more different codons encoding the same amino acid.

21. The method of claim 4, wherein said Cas9 protein comprises an activity portion having an amino acid sequence that is modified compared to an amino acid sequence of a corresponding wild-type Cas9 protein and cleaves only one strand of DNA, wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.

22. The method of claim 21, wherein the Cas9 protein is transcribed and translated from a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, wherein the nucleotide sequence is modified relative to a corresponding wild-type nucleotide sequence, wherein the modification replaces one or more codons in the wild-type nucleotide sequence with one or more different codons encoding the same amino acid.

23. The method of claim 21, wherein the single molecule DNA-targeting RNA is transcribed from a vector comprising a nucleotide sequence encoding the single molecule DNA-targeting RNA.

24. The method of claim 23, wherein the nucleotide sequence is operably linked to a control element operable in a desired cell type.

25. The method of claim 23, wherein the vector is selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors.

26. The method of claim 25, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors.

27. The method of claim 21, wherein the Cas9 protein is transcribed and translated from a vector comprising a nucleotide sequence encoding the Cas9 protein.

28. The method of claim 27, wherein the nucleotide sequence is operably linked to a control element operable in a desired cell type.

29. The method of claim 27, wherein the vector is selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors.

30. The method of claim 29, wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors.

31. The method of claim 21, wherein the CRISPR-Cas system comprises two or more single molecule DNA-targeting RNAs.

32. The method of claim 21, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule.

33. The method of claim 21, wherein the method comprises editing the target DNA molecule by a homology-directed repair mechanism.

34. The method of claim 21, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).

35. The method of claim 34, wherein the one or more PTD(s) aid in traversal of an organelle membrane.

36. The method of claim 34, wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

268 and 269.

37. The method of claim 4, wherein said Cas9 protein comprises an activity portion having an amino acid sequence that is modified compared to an amino acid sequence of a corresponding wild-type Cas9 protein.

38. The method of claim 4, wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:

433.

39. The method of claim 4, wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule.

40. An engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising

a) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein;

and

b) a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA;

wherein the single molecule DNA-targeting RNA comprises;

i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and

ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,

wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides; and

wherein the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule, whereby said system is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule.

41. The system of claim 40, wherein one or both of the nucleic acids of a) and b) are one or more vectors and wherein the nucleotide sequence encoding said Cas9 protein and/or the nucleotide sequence encoding said single molecule DNA-targeting RNA are operably linked to a control element operable in a desired cell type.

42. The system of claim 41, wherein the one or more vectors are selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors.

43. The system of claim 42, wherein said viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors.

44. The system of claim 40, comprising two or more single molecule DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs.

45. The system of claim 40, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).

46. The system of claim 45, wherein the one or more PTD(s) aid in traversal of an organelle membrane.

47. The system of claim 45, wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

268 and 269.

48. The system of claim 40, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.

49. The system of claim 40, wherein the nucleotide sequence encoding said Cas9 protein is modified relative to a corresponding wild-type nucleotide sequence, wherein the modification replaces one or more codons in the wild-type nucleotide sequence with one or more different codons encoding the same amino acid.

50. The system of claim 40, wherein said Cas9 protein comprises an activity portion having an amino acid sequence that is modified compared to an amino acid sequence of a corresponding wild-type Cas9 protein and is capable of cleaving only one strand of DNA, wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.

51. The system of claim 50, wherein the nucleotide sequence encoding said Cas9 protein is modified relative to a corresponding wild-type nucleotide sequence, wherein the modification replaces one or more codons in the wild-type nucleotide sequence with one or more different codons encoding the same amino acid.

52. The system of claim 50, comprising two or more single molecule DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs.

53. The system of claim 50, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)).

54. The system of claim 53, wherein the one or more PTD(s) aid in traversal of an organelle membrane.

55. The system of claim 53, wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

268 and 269.

56. The system of claim 40, wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:

433.

57. The system of claim 40, wherein the system comprises a donor polynucleotide and the system is capable of editing the target DNA molecule by inserting a sequence of the donor polynucleotide into a cleaved strand of the target DNA molecule.

58. A Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising:

a Cas9 protein; and

a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises;

i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and

ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex of a protein-binding segment,

wherein i) and ii) are arranged in a 5′

to 3′

orientation and are covalently linked to one another with intervening nucleotides;

wherein the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule, and

wherein the single molecule DNA-targeting RNA comprises one or more sequence modifications compared to a sequence of a corresponding wild type tracrRNA and/or crRNA.

59. The Type II CRISPR-Cas system of claim 58, wherein the modification comprises an engineered secondary structure.

60. The Type II CRISPR-Cas system of claim 58, wherein the modification comprises a reduction in the length and/or degree of complementation in a region of hybridization, compared to a region of hybridization of a wild type DNA-targeting RNA, in the portion of the protein-binding segment that forms a double-stranded RNA duplex.

61. The Type II CRISPR-Cas system of claim 58, wherein the protein-binding segment comprises an artificial loop.

62. The Type II CRISPR-Cas system of claim 58, wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:

433.

63. The Type II CRISPR-Cas system of claim 58, wherein the modification comprises adding, removing, or otherwise altering loops and/or hairpins in the single molecule DNA-targeting RNA.

64. The Type II CRISPR-Cas system of claim 58, wherein the modification comprises one or more additional segments at the 5′

end of the targeter-RNA.

65. The Type II CRISPR-Cas system of claim 58, wherein the modification comprises one or more modified nucleotides in the nucleotide sequence.

66. The Type II CRISPR-Cas system of claim 65, wherein the one or more modified nucleotides comprises at least one non-naturally-occurring nucleotide, nucleotide mimetic, or analog thereof.

67. The Type II CRISPR-Cas system of claim 66, wherein the one or more modified nucleotides are modified at the ribose, phosphate, and/or base moiety.

68. The Type II CRISPR-Cas system of claim 66, wherein the one or more modified nucleotides are selected from the group consisting of 2′

-0-methyl analogs or 2′

-fluoro analogs.

69. The Type II CRISPR-Cas system of claim 66, wherein the one or more modified nucleotides are selected from the group consisting of 2-aminopurine, 5-bromo-uridine, pseudouridine, and 7-methylguanosine.

70. A method of cleaving or editing a target DNA molecule or modulating transcription of a gene encoded by the target DNA molecule, the method comprising

contacting the system of claim 58 with a target DNA molecule having the target sequence.

71. The method of claim 70, wherein the target DNA molecule is edited, wherein the editing comprises modifying the target DNA molecule by inserting a sequence of a donor polynucleotide, said insertion resulting in an insertion, deletion, or substitution of one or more nucleotides.