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 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 Assignments
0 Petitions
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.
-
Citations
30 Claims
-
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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
-
-
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. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
Specification