METHODS AND COMPOSITIONS FOR RNA-DIRECTED TARGET DNA MODIFICATION AND FOR RNA-DIRECTED MODULATION OF TRANSCRIPTION
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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.
201 Citations
157 Claims
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1-155. -155. (canceled)
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156. A method of cleaving a nucleic acid comprising contacting a target DNA molecule having a target sequence with
a) a Cas9 protein; - and
b) a single molecule DNA-targeting RNA comprising i) a targeter-RNA that hybridizes with the target sequence, and ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA comprises the nucleotide sequence set forth as SEQ ID NO;
1347,wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides, wherein the single molecule DNA-targeting RNA forms a complex with the Cas9 protein, whereby the single molecule DNA-targeting RNA targets the target sequence, and the Cas9 protein cleaves the target DNA molecule. - View Dependent Claims (157)
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Specification