Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
First Claim
1. A method of targeting and binding a target DNA, the method comprising:
- contacting the target DNA with;
(a) a Cas9 protein that comprises a mutation in a RuvC and/or an HNH domain; 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.
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Accused Products
Abstract
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.
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Citations
30 Claims
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1. A method of targeting and binding a target DNA, the method comprising:
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contacting the target DNA with; (a) a Cas9 protein that comprises a mutation in a RuvC and/or an HNH domain; 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of modifying a target DNA, the method comprising:
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contacting the target DNA with; (a) a Cas9 protein that comprises a mutation in a RuvC and/or an HNH domain; 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 modification of the target DNA. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method of cleaving a target DNA in a cell, the method comprising:
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contacting the target DNA in the cell with; (a) a Cas9 protein that comprises a mutation in a RuvC and/or an HNH domain; 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, wherein the nucleotide sequence that is complementary to the target sequence is 15-25 nucleotides (nt) long; 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 comprises;
introducing into the cell the single molecule DNA-targeting RNA or a nucleic acid that encodes the single molecule DNA-targeting RNA, and introducing into the cell the Cas9 protein or a nucleic acid that encodes the Cas9 protein,wherein said contacting results in cleavage of the target DNA.
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Specification