Compositions and methods of nucleic acid-targeting nucleic acids

Patent metadata

Field	Value
Publication number	US-9803194-B2
Application number	US-201615202518-A
Country	US
Kind code	B2
Filing date	Jul 5, 2016
Priority date	Mar 14, 2013
Publication date	Oct 31, 2017
Grant date	Oct 31, 2017

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 sequence, comprising:
contacting a mixture of DNA fragments comprising the target DNA sequence with a complex comprising an enzymatically inactive site-directed polypeptide encoded by a Cas gene 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 Cas gene is a Type II CRISPR-Cas gene.

3. The method of claim 1 , wherein the site-directed polypeptide comprises an affinity tag, the nucleic acid targeting nucleic acid comprises an affinity tag, or a combination thereof.

4. 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.

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

6. The method of claim 5 , 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.

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

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

9. 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.

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

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

12. The method of claim 11 , 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.

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

14. The method of claim 13 , 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.

15. The method of claim 1 , wherein the nucleic acid-targeting nucleic acid comprises RNA.

16. 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.

17. The method of claim 16 , wherein the tracr nucleic acid comprises the affinity tag.

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

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

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

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

22. The method of claim 21 , 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 Daniel

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

• A61P31/04

  Antibacterial agents · CPC title