Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
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.
1. A method of targeting and binding a target DNA in a prokaryotic cell, the method comprising:
contacting the target DNA inside of the prokaryotic cell with:
(a) a Cas9 protein; and
(b) a single molecule DNA-targeting RNA comprising, in 5′ to 3′ order:
(i) a targeter-RNA comprising a nucleotide sequence that is complementary to, and hybridizes with, a target sequence of the target DNA; and
(ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex,
wherein (i) and (ii) are covalently linked by intervening nucleotides,
wherein the Cas9 protein and the single molecule DNA-targeting RNA form a complex that binds to the target DNA.
2. The method of claim 1 , wherein the prokaryotic cell is a bacterial cell.
3. The method of claim 1 , wherein the nucleotide sequence that is complementary to the target sequence of the target DNA is 15-25 nucleotides (nt) long.
4. The method of claim 1 , wherein the Cas9 protein is fused to a heterologous polypeptide.
5. The method of claim 1 , wherein the Cas9 protein is fused to a heterologous polypeptide that comprises one or more of: a protein tag, an IF2 domain, a GST domain, a GRPE domain, a 6×His tag, a hemagglutinin (HA) tag, and green fluorescent protein.
6. The method of claim 1 , wherein the single molecule DNA-targeting RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.
7. The method of claim 1 , wherein the single molecule DNA-targeting RNA comprises one or more of: a phosphorothioate, an inverted polarity linkage, an abasic nucleoside linkage, a locked nucleic acid (LNA), a 2′-substituted sugar moiety, a 2′-O-methoxyethyl modified sugar moiety, a 2′-O-methyl modified sugar moiety, a 2′-O-(2-methoxyethyl) modified sugar moiety, a 2′-fluoro modified sugar moiety, a 2′-dimethylaminooxyethoxy modified sugar moiety, a 2′-dimethylaminoethoxyethoxy modified sugar moiety, a peptide nucleic acid (PNA), a morpholino nucleic acid, and a cyclohexenyl nucleic acid (CeNA).
8. The method of claim 1 , wherein the single molecule DNA-targeting RNA comprises a heterologous moiety.
9. The method of claim 1 , wherein the single molecule DNA-targeting RNA comprises one or more of the following heterologous moieties: an acridine; a fluorescein; a rhodamine; a coumarin; and a dye.
10. The method of claim 1 , wherein the activator-RNA comprises the 67 nt tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU UUU (SEQ ID NO: 432) or the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 397).
11. The method of claim 1 , wherein the method comprises contacting the target DNA with two or more single molecule DNA-targeting RNAs, wherein the two or more single molecule DNA-targeting RNAs hybridize to different target sequences within the same or different target DNA molecules.
12. The method of claim 1 , wherein the method comprises introducing the Cas9 protein, an RNA molecule that encodes the Cas9 protein, or a DNA molecule that encodes the Cas9 protein into the prokaryotic cell.
13. The method of claim 1 , wherein the method comprises introducing a nucleic acid encoding the single molecule DNA-targeting RNA into the prokaryotic cell.
14. The method of claim 1 , wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
15. A method of cleaving a target DNA in a prokaryotic cell, the method comprising:
contacting the target DNA inside of the prokaryotic cell with:
(a) a Cas9 protein; and
(b) a single molecule DNA-targeting RNA comprising, in 5′ to 3′ order:
(i) a targeter-RNA comprising a nucleotide sequence that is complementary to, and hybridizes with, a target sequence of the target DNA; and
(ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex,
wherein (i) and (ii) are covalently linked by intervening nucleotides,
wherein said contacting results in cleavage of the target DNA.
16. The method of claim 15 , wherein the prokaryotic cell is a bacterial cell.
17. The method of claim 15 , wherein the nucleotide sequence that is complementary to the target sequence of the target DNA is 15-25 nucleotides (nt) long.
18. The method of claim 15 , wherein the Cas9 protein is fused to a heterologous polypeptide.
19. The method of claim 15 , wherein the Cas9 protein comprises one or more of: a protein tag, an IF2 domain, a GST domain, a GRPE domain, a 6×His tag, a hemagglutinin (HA) tag, and green fluorescent protein.
20. The method of claim 15 , wherein the single molecule DNA-targeting RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.
21. The method of claim 15 , wherein the single molecule DNA-targeting RNA comprises a non-natural internucleoside linkage and a modified sugar moiety.
22. The method of claim 15 , wherein the single molecule DNA-targeting RNA comprises one or more of: a phosphorothioate, an inverted polarity linkage, an abasic nucleoside linkage, a locked nucleic acid (LNA), a 2′-substituted sugar moiety, a 2′-O-methoxyethyl modified sugar moiety, a 2′-O-methyl modified sugar moiety, a 2′-O-(2-methoxyethyl) modified sugar moiety, a 2′-fluoro modified sugar moiety, a 2′-dimethylaminooxyethoxy modified sugar moiety, a 2′-dimethylaminoethoxyethoxy modified sugar moiety, a peptide nucleic acid (PNA), a morpholino nucleic acid, and a cyclohexenyl nucleic acid (CeNA).
23. The method of claim 15 , wherein the single molecule DNA-targeting RNA comprises a heterologous moiety.
24. The method of claim 15 , wherein the single molecule DNA-targeting RNA comprises one or more of the following heterologous moieties: an acridine; a fluorescein; a rhodamine; a coumarin; and a dye.
25. The method of claim 15 , wherein the activator-RNA comprises the 67 nt tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU UUU (SEQ ID NO: 432) or the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 397).
26. The method of claim 15 , wherein the method comprises contacting the target DNA with two or more single molecule DNA-targeting RNAs, wherein the two or more single molecule DNA-targeting RNAs hybridize to different target sequences within the same or different target DNA molecules.
27. The method of claim 15 , wherein the method comprises introducing the Cas9 protein, an RNA molecule that encodes the Cas9 protein, or a DNA molecule that encodes the Cas9 protein into the prokaryotic cell.
28. The method of claim 15 , wherein the method comprises introducing a nucleic acid encoding the single molecule DNA-targeting RNA into the prokaryotic cell.
29. The method of claim 15 , wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.
30. The method of claim 15 , comprising introducing a donor polynucleotide into the prokaryotic cell.