RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX

• US 20190085329A1
• Filed: 10/01/2018
• Published: 03/21/2019
• Est. Priority Date: 03/20/2012
• Status: Active Grant

• Current Assignee: Vilnius University
• Original Assignee: Vilnius University
• Inventors: {hacek over (S)}IK{hacek over (S)}NYS, Virginijus, Gasiunas, Giedrius, Karvelis, Tautvydas
• Granted Patent: US 10,844,378 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/102 Mutagenizing 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 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• 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 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2320/00 Applications; Uses
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule using an RNA-guided DNA endonuclease comprising at least one RNA sequence and at least one clan RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro or using a cassette containing a single repeat-spacer-repeat unit.

Claims

1-41. -41. (canceled)

42. A method of preparing a Cas9-crRNA complex for modification of a target DNA molecule, the method comprising:

contacting a Cas9 polypeptide with a crRNA and a tracrRNA in vitro to form the Cas9-crRNA complex, wherein the crRNA is engineered to site-specifically bind to a polynucleotide sequence of the target DNA molecule, the crRNA comprising a polynucleotide sequence complementary to the polynucleotide sequence of the target DNA molecule.

43. The method of claim 42, further comprising:

contacting the Cas9-crRNA complex with the target DNA molecule, whereby the Cas9-crRNA complex site-specifically binds to the target DNA molecule.

44. The method of claim 43, wherein the target DNA molecule is double stranded.

45. The method of claim 42, wherein the Cas9 polypeptide is produced by recombinant DNA technology or chemical synthesis.

46. The method of claim 42, wherein the Cas9 polypeptide is isolated from a genetically modified microorganism.

47. The method of claim 42, further comprising:

contacting the Cas9-crRNA complex with the target DNA molecule, whereby the Cas9-crRNA complex site-specifically cleaves the target DNA molecule.

48. The method of claim 42, wherein the polynucleotide sequence of the crRNA complementary to the polynucleotide sequence of the target DNA molecule comprises about 20 nucleotides.

49. The method of claim 42, further comprising producing the crRNA by chemical synthesis.

50. The method of claim 42, wherein the Cas9-crRNA complex is formed in the absence of RNaseIII.

51. The method of claim 42, wherein the Cas9 polypeptide comprises a mutation in a RuvC active site motif or a HNH active site motif of the Cas9 polypeptide.

52. A method of preparing a Cas9-crRNA complex for modification of a heterologous target DNA molecule, the method comprising:

contacting a Cas9 polypeptide with a crRNA and a tracrRNA in vitro to form the Cas9-crRNA complex, wherein the crRNA is heterologous to the Cas9 polypeptide and engineered to bind to the Cas9-crRNA complex to the target DNA molecule; and

contacting the target DNA molecule with the Cas9-crRNA complex, whereby the Cas9-crRNA complex modifies the target DNA molecule.

53. The method of claim 52, wherein the crRNA is obtained by:

identifying a polynucleotide sequence of the target DNA molecule; and

preparing the crRNA, such that the crRNA includes a polynucleotide sequence complementary to the polynucleotide sequence of the DNA molecule.

54. The method of claim 52, wherein the crRNA is prepared by chemical synthesis.

55. The method of claim 52, wherein the Cas9 polypeptide comprises a mutation in a RuvC active site motif or a HNH active site motif of the Cas9 polypeptide.

56. The method of claim 52, wherein the crRNA includes a polynucleotide sequence of at least 20 nucleotides complementary to a polynucleotide sequence of the target DNA molecule.

57. The method of claim 52, wherein the Cas9 polypeptide is contacted with the crRNA and the tracrRNA in the absence of RNaseIII.

58. The method of claim 52, wherein the target DNA molecule includes a protospacer-adjacent motif.

59. The method of claim 52, wherein the target DNA molecule is double stranded, and wherein the Cas9-crRNA complex modifies the target DNA molecule by site-specific double stranded cleavage of the target DNA molecule.

60. A method of preparing a Cas9-crRNA complex for modification of a target DNA molecule, the method comprising:

identifying a polynucleotide sequence of the target DNA molecule;

preparing a crRNA having a polynucleotide sequence complementary to the polynucleotide sequence of the DNA molecule; and

contacting a Cas9 polypeptide with the crRNA and a tracrRNA to form the Cas9-crRNA complex, wherein the crRNA is engineered to site-specifically bind to the target DNA molecule and is heterologous to the Cas9 polypeptide.

61. The method of claim 60, wherein the Cas9 polypeptide comprises a mutation in a RuvC active site motif or a HNH active site motif of the Cas9 polypeptide.

62. The method of claim 60, wherein the Cas9 polypeptide is isolated from a genetically modified microorganism, or wherein the Cas9 polypeptide is produced by recombinant DNA technology or chemical synthesis.

63. The method of claim 60, further comprising:

contacting the target DNA molecule with the Cas9-crRNA complex, whereby the Cas9-crRNA complex cleaves the target DNA molecule.

64. The method of claim 63, wherein the crRNA is heterologous to the target DNA molecule.

65. A method of preparing a Cas9-crRNA complex for modification of a target DNA molecule, the method comprising:

contacting a recombinant Cas9 polypeptide with a crRNA and a tracrRNA to form the Cas9-crRNA complex, wherein the crRNA is programmed to site-specifically bind to the target DNA molecule, and wherein the crRNA is heterologous to the Cas9 polypeptide and heterologous to the target DNA molecule; and

contacting the target DNA molecule with the Cas9-crRNA complex, whereby the Cas9-crRNA complex site-specifically modifies the target DNA molecule.

66. The method of claim 65, wherein the Cas9 polypeptide comprises a mutation in a RuvC active site motif or a HNH active site motif, whereby the Cas9 polypeptide is a nickase;

and wherein the Cas9-crRNA complex site-specifically modifies the target DNA molecule by nicking the target DNA molecule.

67. The method of claim 65, wherein the Cas9-crRNA complex is formed in the absence of RNaseIII.