Compositions and methods of nucleic acid-targeting nucleic acids

Patent metadata

Field	Value
Publication number	US-2016046978-A1
Application number	US-201514751055-A
Country	US
Kind code	A1
Filing date	Jun 25, 2015
Priority date	Mar 14, 2013
Publication date	Feb 18, 2016
Grant date	—

Abstract

This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof.

First claim

1 - 26 . (canceled)

27 . A method for identifying off-target binding sites relative to a target DNA target sequence, comprising:
contacting a mixture of DNA fragments comprising the target DNA sequence with an enzymatically inactive Cas9 protein in a complex with a nucleic acid targeting nucleic acid comprising a crRNA and a tracrRNA, wherein (i) the Cas9 protein comprises an affinity tag, the nucleic acid targeting nucleic acid comprises an affinity tag, or a combination thereof, and (ii) the Cas9 in the complex with the nucleic acid targeting nucleic acid binds to the target DNA sequence; isolating the Cas9 protein in the complex with the nucleic acid targeting nucleic acid and bound DNA fragments using a capture agent that binds to the affinity tag; purifying the DNA fragments that were bound by the Cas9 in the complex with the nucleic acid targeting nucleic acid; sequencing the DNA fragments that were bound by the Cas9 in the complex with the nucleic acid targeting nucleic acid; and identifying the off-target binding sites from the sequences of the DNA fragments that were bound by the Cas9 in the complex with the nucleic acid targeting nucleic acid that did not contain the target DNA binding sequence.

Assignees

• Caribou Biosciences Inc

Inventors

• May Andrew Paul
• Haurwitz Rachel E
• Doudna Jennifer A
• Berger James M
• Carter Matthew Merrill
• Donohoue Paul

Classifications

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P9/10

  for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis · CPC title

• A61P27/02

  Ophthalmic agents · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title

• A61P35/00

  Antineoplastic agents · CPC title