RNA-DIRECTED DNA CLEAVAGE AND GENE EDITING BY CAS9 ENZYME FROM NEISSERIA MENINGITIDIS

• US 20140349405A1
• Filed: 05/22/2014
• Published: 11/27/2014
• Est. Priority Date: 05/22/2013
• Status: Abandoned Application

• Current Assignee: Northwestern University, Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Northwestern University, Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Sontheimer, Erik J., Thomson, James, Hou, Zhonggang, Zhang, Yan, Mondragon, Alfonso, Rajan, Rakhi
• Application Number:
  US14/285,252
• Publication Number:
  US 20140349405A1
• Time in Patent Office: Days
• US Class Current: 435/462
• CPC Class Codes: C07K 14/22 from Neisseriaceae (F)
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• 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 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed are components and methods for RNA-directed DNA cleavage and gene editing. The components include and the methods utilize a Cas9 protein from Neisseria and one or more RNA molecules in order to direct the Cas9 protein to bind to and optionally cleave or nick a target sequence.

Claims

1. A method for modifying a target DNA sequence in a cell, the method comprising:

(a) expressing a Cas9 protein from a Neisseria species or a variant protein thereof in the cell; and

(b) expressing or transfecting an RNA in the cell wherein the RNA binds to the Cas9 protein or variant, and the RNA hybridizes to the target DNA sequence.

2. The method of claim 1, wherein the Cas9 protein or variant protein has nuclease activity and cleaves both strands of the target DNA sequence.

3. The method of claim 2, further comprising contacting the target DNA sequence with a homologous DNA fragment.

4. The method of claim 1, wherein the Cas9 protein or variant protein has nuclease activity and nicks a single strand of the target DNA sequence.

5. The method of claim 4, further comprising contacting the target DNA sequence with a homologous DNA fragment.

6. The method of claim 1, wherein the Cas9 protein or variant protein has no nuclease activity and binds to the target sequence.

7. The method of claim 1, wherein the Cas9 protein or variant protein is expressed from a nucleic acid having a codon sequence that is optimized for expression in the cell.

8. The method of claim 1, wherein the variant has an amino acid sequence that is at least 80% identical to a Cas9 protein from a Neisseria species.

9. The method of claim 1, wherein the Cas9 protein is from Neisseria meningitidis.

10. The method of claim 1, wherein expressing a Cas9 protein from a Neisseria species or a variant protein thereof in the cell comprises transfecting the cell with an expression vector that expresses the Cas9 protein from a eukaryotic promoter.

11. The method of claim 1, wherein expressing a Cas9 protein from a Neisseria species or a variant protein thereof in the cell comprises transfecting the cell with an mRNA that encodes the Cas9 protein.

12. The method of claim 1, wherein expressing an RNA in the cell that binds to the Cas9 protein or variant and hybridizes to the target DNA sequence comprises transfecting the cell with an expression vector that expresses the RNA from a eukaryotic promoter.

13. The method of claim 1, wherein the cell is a prokaryotic cell.

14. The method of claim 1, wherein the cell is a eukaryotic cell.

15. The method of claim 1, wherein the cell is a stem cell.

16. The method of claim 1, wherein the cell is an embryonic stem cell.

17. The method of claim 1, wherein the cell is an induced pluripotent stem cell.

18. The method of claim 1, wherein the RNA comprises two molecules of duplexed RNA.

19. The method of claim 1, wherein the RNA comprises a single RNA molecule forming a hairpin structure.

20. The method of claim 1, wherein the RNA comprises an RNA mimic of green fluorescent protein (GFP).

21. The method of claim 1, further comprising contacting the target DNA sequence with 4-hydroxybenzylidene, 3,5-dimethoxy-4-hydroxybenzylidene, or a 3,5-difluoro-4-hydroxybenzylidene.

22. The method of claim 1, wherein the RNA comprises Xist RNA.

27. A kit for performing the method of claim 1 comprising:

(a) a vector for expressing a Cas9 protein from a Neisseria species or a variant protein thereof in the cell; and

(b) a vector for expressing an RNA in the cell, wherein the RNA binds to the Cas9 protein or variant, and the RNA hybridizes to the target DNA sequence.

23. A recombinant Cas9 protein from a Neisseria species or a variant thereof comprising a nuclear localization signal.

25. A polynucleotide encoding the protein of claim 23.

26. A cell transfected with the polynucleotide of claim 25.

28. A kit comprising the protein of claim 23.

29. A kit comprising the polynucleotide of claim 25.

30. A kit comprising the cell of claim 26.

24. A recombinant Cas9 protein from a Neisseria species or a variant thereof comprising a ligand or a tag for purifying or identifying the Cas9 protein.