Compostions and methods of nucleic acid-targeting nucleic acids

Patent metadata

Field	Value
Publication number	US-9410198-B2
Application number	US-201514751055-A
Country	US
Kind code	B2
Filing date	Jun 25, 2015
Priority date	Mar 14, 2013
Publication date	Aug 9, 2016
Grant date	Aug 9, 2016

Abstract

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

First claim

What is claimed is:

1. 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 a complex comprising an enzymatically inactive Cas9 protein and a nucleic acid-targeting nucleic acid, wherein (i) the complex comprises an affinity tag, and (ii) the complex binds to the target DNA sequence;
purifying the DNA fragments bound by the complex using the affinity tag; and
identifying the off-target binding sites based on the DNA fragments bound by the complex that do not comprise the target DNA binding sequence.

2. The method of claim 1 , wherein the Cas9 protein comprises an affinity tag, the nucleic acid targeting nucleic acid comprises an affinity tag, or a combination thereof.

3. The method of claim 1 , wherein the purifying further comprises isolating the complex and the DNA fragments bound by the complex using a capture agent that binds to the affinity tag.

4. The method of claim 1 , wherein the affinity tag is a nucleic acid sequence.

5. The method of claim 4 , wherein the nucleic acid sequence is selected from the group consisting of an RNA-binding protein binding sequence, a DNA-binding protein binding sequence, a sequence hybridizable to an affinity-tagged polynucleotide, a synthetic RNA aptamer, a synthetic DNA aptamer, and a combination thereof.

6. The method of claim 1 , wherein the affinity tag is a small molecule.

7. The method of claim 6 , wherein the small molecule is selected from the group consisting of biotin, digitoxin, a fluorescent molecule, and a combination thereof.

8. The method of claim 1 , wherein the identifying further comprises sequencing the DNA fragments bound by the complex that do not comprise the target DNA binding sequence.

9. The method of claim 1 , wherein the complex further comprises a detectable label.

10. The method of claim 1 , wherein the affinity tag is a detectable label.

11. The method of claim 10 , wherein the identifying further comprises contacting an array of immobilized polynucleotides with the DNA fragments bound by the complex that do not comprise the target DNA binding sequence.

12. The method of claim 1 , wherein the DNA fragments of the mixture of DNA fragments comprise a detectable label.

13. The method of claim 12 , wherein the identifying further comprises contacting an array of immobilized polynucleotides with the DNA fragments bound by the complex that do not comprise the target DNA binding sequence.

14. The method of claim 1 , wherein the nucleic acid-targeting nucleic acid is a double-guide nucleic acid-targeting nucleic acid comprising a spacer nucleic acid and a tracr nucleic acid.

15. The method of claim 14 , wherein the tracr nucleic acid comprises the affinity tag.

16. The method of claim 14 , wherein the tracr nucleic acid comprises RNA.

17. The method of claim 16 , wherein the tracr nucleic acid further comprises DNA.

18. The method of claim 14 , wherein the tracr nucleic acid comprises DNA.

19. The method of claim 1 , wherein the mixture of DNA fragments is obtained from genomic DNA of a cell.

20. The method of claim 19 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an algal cell, a seaweed cell, a fungal cell, an animal cell, a cell from an invertebrate animal, a cell from a vertebrate animal, and a cell from a mammal.

Assignees

• Caribou Biosciences Inc

Inventors

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

Classifications

• 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

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P29/00

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

• A61P31/04

  Antibacterial agents · CPC title

• A61P27/02

  Ophthalmic agents · CPC title