RNA-directed DNA cleavage by the Cas9-crRNA complex

• US 10,844,378 B2
• Filed: 10/01/2018
• Issued: 11/24/2020
• 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
• Primary Examiner(s): Leavitt, Maria G
• Application Number:
  US16/148,783
• Publication Number:
  US 20190085329A1
• Time in Patent Office: 785 Days
• Field of Search: None
• 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 of an 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. A method of preparing a Cas9-crRNA complex with engineered specificity towards a target DNA molecule, the method comprising:

engineering a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) to site-specifically bind to a polynucleotide sequence of the target DNA molecule, wherein the engineering includes re-programming a polynucleotide sequence of the crRNA by generating the polynucleotide sequence of the engineered crRNA complementary to the polynucleotide sequence of the target DNA molecule; and

contacting a CRISPR associated polypeptide 9 (Cas9 polypeptide) with the engineered crRNA and a trans-activating RNA (tracrRNA) in vitro to form the Cas9-crRNA complex, the Cas9-crRNA complex having engineered specificity towards a site of the target DNA molecule for modifying the target DNA molecule.

2. The method of claim 1, wherein the Cas9 polypeptide is produced by recombinant DNA technology or chemical synthesis.

3. The method of claim 1, wherein the Cas9 polypeptide is isolated from a genetically modified microorganism.

4. The method of claim 1, wherein the polynucleotide sequence of the engineered crRNA complementary to the polynucleotide sequence of the target DNA molecule comprises about 20 nucleotides.

5. The method of claim 1, wherein engineering the crRNA is performed by chemical synthesis.

6. The method of claim 1, wherein the Cas9-crRNA complex is formed in the absence of ribonuclease III (RNaseIII).

7. The method of claim 1, wherein the Cas9 polypeptide comprises a mutation in a RuvC active site motif or a HNH active site motif of the Cas9 polypeptide.

8. A method of preparing a Cas9-crRNA complex, the method comprising:

contacting a clustered regularly interspaced short palindromic repeats (CRISPR) associated polypeptide 9 (Cas9 polypeptide) with an engineered CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA) in vitro to form the Cas9-crRNA complex, wherein the engineered crRNA is generated to guide the Cas9-crRNA complex to a region comprising a site in a target DNA molecule, so that the Cas9-crRNA complex binds to the target DNA molecule, and wherein the engineered crRNA is not generated through processing of a bacterial CRISPR repeat-spacer array.

9. The method of claim 8, wherein the engineered crRNA is obtained by:

identifying a polynucleotide sequence of the target DNA molecule; and

generating the engineered crRNA by re-programming a polynucleotide sequence of a crRNA to be complementary to the polynucleotide sequence of the DNA molecule.

10. The method of claim 8, wherein the engineered crRNA is generated by chemical synthesis.

11. The method of claim 8, wherein the Cas9 polypeptide comprises a mutation in a RuvC active site motif or a HNH active site motif of the Cas9 polypeptide.

12. The method of claim 8, wherein the engineered crRNA includes a polynucleotide sequence of at least 20 nucleotides complementary to a polynucleotide sequence of the target DNA molecule.

13. The method of claim 8, wherein the Cas9 polypeptide is contacted with the engineered crRNA and the tracrRNA in the absence of ribonuclease III (RNaseIII).

14. A method of preparing a Cas9-crRNA complex, the method comprising:

identifying a polynucleotide sequence of a target DNA molecule;

engineering a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) having a polynucleotide sequence, wherein the engineering includes re-programming the polynucleotide sequence of the crRNA to be complementary to the polynucleotide sequence of the DNA molecule and to site-specifically bind to the target DNA molecule; and

contacting a CRISPR associated polypeptide 9 (Cas9 polypeptide) with the engineered crRNA and a trans-activating RNA (tracrRNA) in vitro to form the Cas9-crRNA complex.

15. The method of claim 14, wherein the Cas9 polypeptide comprises a mutation in a RuvC active site motif or a HNH active site motif of the Cas9 polypeptide.

16. The method of claim 14, 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.

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

contacting a recombinant clustered regularly interspaced short palindromic repeats (CRISPR) associated polypeptide 9 (Cas9 polypeptide) with an engineered CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA) in vitro to form the Cas9-crRNA complex, wherein the engineered crRNA is re-programmed by generating a sequence of the engineered crRNA complementary to a polynucleotide sequence of the target DNA molecule to site-specifically bind to the target DNA molecule, and wherein the engineered crRNA is not generated through processing of a bacterial CRISPR repeat-spacer array.

18. The method of claim 17, 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.

19. The method of claim 17, wherein the Cas9-crRNA complex is formed in the absence of ribonuclease III (RNaseIII).