NUCLEIC ACID ENRICHMENT USING CAS9

• US 20140356867A1
• Filed: 05/29/2014
• Published: 12/04/2014
• Est. Priority Date: 05/29/2013
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Peter, Brian Jon, Ach, Robert A.
• Granted Patent: US 11,414,695 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method of enriching for a fragment of a genome, as well as corresponding compositions and kits, are provided. In certain embodiments, the method comprises: (a) contacting a sample comprising fragmented DNA with a Cas9-gRNA complex comprising mutant Cas9 protein that has inactivated nuclease activity and a Cas9-associated guide RNA that is complementary to a site in the DNA, to produce a Cas9-fragment complex that comprises a fragment of the fragmented DNA; and (b) isolating the complex. In addition, other methods and compositions for Cas9/CRISPR-mediated nucleic acid manipulation are also provided.

Claims

1. A method comprising:

(a) contacting a sample comprising fragmented genomic DNA with a Cas9-gRNA complex comprising a mutant Cas9 protein that has inactivated nuclease activity and a Cas9-associated guide RNA that is complementary to a site in said genomic DNA, to produce a Cas9-fragment complex that comprises a fragment of the fragmented genomic DNA; and

(b) isolating said Cas9-fragment complex.

2. The method of claim 1, wherein the Cas9 protein comprises a mutant Cas9 that has amino acid substitutions which affect DNA binding, DNA cleavage, or gRNA binding.

3. The method of claim 2, wherein the Cas9 protein has amino acid substitutions at D10 and H840, or sites corresponding thereto.

4. The method of claim 1, wherein the fragment is greater than 10 kb in length.

5. The method of claim 1, wherein said isolating is done using a capture agent that specifically binds to said Cas9 protein.

6. The method of claim 5, wherein said capture agent is an antibody.

7. The method of claim 1, wherein said Cas9-associated guide RNA comprises an affinity tag, and said isolating is done using a capture agent for said affinity tag.

8. The method of claim 1, wherein the fragmented genomic DNA is made by fragmenting a genomic sample using Cas9.

9. The method of claim 1, wherein said Cas9 protein is a fusion with an affinity tag, and said isolating is done using a capture agent for the affinity tag.

10. The method of claim 1, wherein said contacting comprises contacting said fragmented genomic DNA with a Cas9 protein and a set of Cas9-associated guide RNAs that each comprise sequences complementary to different sites in the genomic DNA.

11. The method of claim 10, wherein the different sites are in a defined region in the range of 1 kb to 200 kb.

12. The method of claim 10, wherein the different sites to which said Cas9-associated guide RNAs bind are distributed along a defined region.

13. The method of claim 1, further comprising:

(c) releasing the fragment from the isolated Cas9-fragment complex of (b).

14. The method of claim 13, further comprising genotyping the released fragment.

15. The method of claim 14, wherein the genotyping is done by sequencing.

16. The method of claim 13, wherein the released fragment is a double-stranded fragment.

17. The method of claim 13, further comprising (d) amplifying the released fragments to produce PCR products.

18. The method of claim 15, further comprising sequencing the PCR products.

19. The method of claim 1, wherein the sample comprises a pool of fragmented genomic DNA from a plurality of samples, wherein the fragments in the sample have a molecular barcode to indicate their source.

20. A composition comprising a mutant Cas9 protein that has inactivated nuclease activity and a plurality of Cas9-associated guide RNA that are complementary to different sites in genomic DNA.