Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
First Claim
1. A composition comprising(a) a Cas9 protein or a nucleic acid encoding the Cas9 protein;
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(i) a targeter-RNA that is capable of hybridizing with a target sequence of a target DNA, and(ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex,wherein (i) and (ii) are covalently linked,wherein the DNA-targeting RNA is capable of forming a complex with the Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA.
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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 composition comprising
(a) a Cas9 protein or a nucleic acid encoding the Cas9 protein; - and
(b) a DNA-targeting RNA comprising; (i) a targeter-RNA that is capable of hybridizing with a target sequence of a target DNA, and (ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex, wherein (i) and (ii) are covalently linked, wherein the DNA-targeting RNA is capable of forming a complex with the Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A kit comprising
(a) a Cas9 protein or a nucleic acid encoding the Cas9 protein; - and
(b) a DNA-targeting RNA comprising; (i) a targeter-RNA that is capable of hybridizing with a target sequence of a target DNA, and (ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex, wherein (i) and (ii) are covalently linked, wherein the DNA-targeting RNA is capable of forming a complex with the Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA; and wherein (a) and (b) are separated. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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Specification