RNA-directed DNA cleavage by the Cas9-crRNA complex

• US 9,637,739 B2
• Filed: 03/15/2013
• Issued: 05/02/2017
• Est. Priority Date: 03/20/2012
• Status: Active Grant

• Current Assignee: Vilnius University
• Original Assignee: Vilnius University
• Inventors: Karvelis, Tautvydas, {hacek over (S)}ik{hacek over (s)}nys, Virginijus, Gasiunas, Giedrius
• Primary Examiner(s): Schultz, Doug
• Application Number:
  US14/385,241
• Publication Number:
  US 20150045546A1
• Time in Patent Office: 1,509 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 for site-specific modification of a target DNA molecule, the method comprising

assembling a recombinant Cas9-crRNA complex in vitro by combining a Cas9 protein, an engineered crRNA, and a tracrRNA under conditions suitable for formation of the complex, and

contacting a target DNA molecule with the recombinant Cas9-crRNA complex, wherein the engineered crRNA is capable of universal targeting and programmed to guide the recombinant Cas9-crRNA complex to a region comprising a site in the target DNA molecule, wherein the crRNA sequence is reprogrammed to be heterologous to the Cas9 protein, and

wherein the site-specific modification of the target DNA molecule is cleavage of the target DNA molecule.

2. The method of claim 1 , wherein a fragment of the crRNA is substantially complementary to the target DNA molecule.

3. The method of claim 2, wherein the fragment of the crRNA that is substantially complementary to the target DNA molecule comprises about 20 nucleotides.

4. The method of claim 2, wherein the fragment of the crRNA that is substantially complementary to the target DNA molecules comprises at least 20 nucleotides.

5. The method of claim 1, wherein the Cas9 protein comprises at least one of an RuvC active site motif and an HNH active site motif.

6. The method of claim 1, wherein the crRNA comprises a 3′

and a 5′

region, wherein the 3′

region comprises at least 22 nucleotides of a CRISPR repeat and the 5′

region comprises at least 20 nucleotides of a spacer sequence engineered to be substantially complementary to the region of the target DNA.

7. The method of claim 1, wherein the target DNA molecule comprises a proto-spacer adjacent motif (PAM) sequence.

8. The method of claim 7, wherein the PAM sequence comprises a nucleic acid molecule having the nucleic acid sequence 5′

-NGGNG.

9. The method of claim 5, wherein one of the RuvC active site motif or the HNH active site motif of the Cas9 protein is inactivated, and wherein the target DNA is double stranded and the modification of the target DNA molecule is site-specific nicking of a single strand of the target DNA molecule.

10. The method of claim 1, wherein the target DNA is double stranded or single stranded.

11. The method of claim 7, wherein the PAM sequence comprises a proto-spacer upstream of the PAM.

12. The method of claim 1, wherein the recombinant Cas9-crRNA complex remains associated with the target DNA molecule after cleavage.

13. The method of claim 10, wherein the target DNA is double stranded and the proto-spacer adjacent motif (PAM) sequence is recognized in the context of double stranded DNA.

14. The method of claim 9, wherein the RuvC active site motif or the HNH active site motif of the Cas9 protein is inactivated by a point mutation.

15. The method of claim 14, wherein the point mutation in the RuvC motif is D31A and the point mutation in the HNH motif is N891A.

16. The method of claim 1 wherein, prior to assembly of the recombinant Cas9-crRNA complex, the engineered crRNA and the tracrRNA are produced by in vitro transcription or chemical synthesis;

and Cas9 protein is produced by a method selected from recombinant DNA technology or chemical synthesis.

17. The method of claim 16 wherein Cas9 protein is isolated from a genetically modified microorganism.

18. The method of claim 1 wherein, prior to assembly of the recombinant Cas9-crRNA complex, Cas9 protein, the engineered crRNA, and the tracrRNA are isolated from a genetically modified microorganism.

19. The method of claim 18 wherein the genetically modified microorganism is created by introducing at least one plasmid encoding Cas9 protein, the engineered crRNA, and the tracrRNA into the microorganism, to result in the genetically modified microorganism.

20. The method of claim 19 wherein Cas9 protein, the engineered crRNA, and the tracrRNA are encoded in two separate plasmids.

21. The method of claim 19 wherein Cas9 protein, the engineered crRNA, and the tracrRNA are encoded in three separate plasmids.

22. The method of claim 1 wherein Cas9 is at least 80% identical with SEQ ID NO:

1.

23. The method of claim 1 wherein the engineered crRNA and the tracrRNA are assembled into a duplex, and then Cas9 protein is incubated with the duplex, to result in the recombinant Cas9-crRNA complex.