METHOD FOR FRAGMENTING GENOMIC DNA USING CAS9

• US 20140357523A1
• 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: Zeiner, Gusti, Peter, Brian Jon, Ach, Robert A., Lindstrom, Derek Lee
• Granted Patent: US 9,873,907 B2
• Time in Patent Office: Days
• US Class Current: 506/11
• 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 for fragmenting a genome is provided. In certain embodiments, the method comprises: (a) combining a genomic sample containing genomic DNA with a plurality of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a Cas9 protein and a set of at least 10 Cas9-associated guide RNAs that are complementary to different, pre-defined, sites in a genome, to produce a reaction mixture; and (b) incubating the reaction mixture to produce at least 5 fragments of the genomic DNA. Also provided is a composition comprising at least 100 Cas9-associated guide RNAs that are each complementary to a different, pre-defined, site in a genome. Kits for performing the method are also provided. In addition, other methods, compositions and kits for manipulating nucleic acids are also provided.

Claims

1. A method for fragmenting a genome comprising:

(a) combining a sample containing genomic DNA with a plurality of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a Cas9 protein and a set of at least 10 Cas9-associated guide RNAs that are complementary to different, pre-defined, sites in a genome, to produce a reaction mixture; and

(b) incubating the reaction mixture to produce at least 5 fragments of the genomic DNA.

2. The method of claim 1, wherein said genomic DNA is mammalian genomic DNA.

3. The method of claim 1, wherein said genomic DNA is human genomic DNA.

4. The method of claim 1, wherein the fragments are all of a defined length.

5. The method of claim 1, wherein said defined length is in the range of 1 kb to 100 kb.

6. The method of claim 5, wherein said defined length is in the range of 1 kb to 10 kb.

7. The method of claim 1, further comprising cloning the fragments into a vector.

8. The method of claim 7, wherein the vector is a fosmid, bac or cosmid vector.

9. The method of claim 1, further comprising sequencing the fragments.

10. The method of claim 1, further comprising amplifying the fragments.

11. The method of claim 1, wherein the at least 10 Cas9-associated guide RNAs comprises at least 100 Cas9-associated guide RNAs that are each complementary to a different, pre-defined, site in said genomic DNA.

12. The method of claim 1, wherein the reaction mixture further comprises a set of Cas9-associated guide RNAs that are complementary to repetitive sequences in said genomic DNA, wherein the incubating results in cleavage of one or more the repetitive sequences targeted by the Cas9-associated guide RNAs.

13. The method of claim 12, wherein the repetitive sequences are cleaved into fragments that range in size from 20 bases to 1 kb.

14. A composition comprising:

a set of at least 10 Cas9-associated guide RNAs that are each complementary to a different, pre-defined sequence.

15. The composition of claim 14, wherein each sequence is a genomic sequence.

16. The composition of claim 14, wherein each sequence is a human genomic sequence.

17. The composition of claim 14, wherein each sequence is a genomic sequence from a different pathogen.

18. The composition of claim 14, further comprising a Cas9 nuclease.

19. The composition of claim 14, wherein said RNAs are in solution.

20. The composition of claim 14, wherein said RNAs are tethered to a substrate in an array.