Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

Patent metadata

Field	Value
Publication number	US-2016130608-A1
Application number	US-201514685502-A
Country	US
Kind code	A1
Filing date	Apr 13, 2015
Priority date	May 25, 2012
Publication date	May 12, 2016
Grant date	—

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.

First claim

1 .- 565 . (canceled)

566 . 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, wherein the targeter-RNA comprises the nucleotide sequence set forth as SEQ ID NO:1348, 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 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.

567 . The method of claim 566 , wherein said double-stranded RNA duplex has a length of from 8 base pairs (bp) to 30 bp.

Assignees

• Charpentier Emmanuelle
• Univ California
• Univ Vienna

Inventors

• Doudna Jennifer A
• Jinek Martin
• Charpentier Emmanuelle
• Chylinski Krzysztof
• Doudna Cate James Harrison
• Lim Wendell
• Qi Lei

Classifications

• H10P14/6512

  by exposure to a gas or vapour · CPC title

• H10P14/20

  of semiconductor materials · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P31/00

  Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics · CPC title