Method for fragmenting genomic DNA using CAS9

Patent metadata

Field	Value
Publication number	US-9873907-B2
Application number	US-201414290896-A
Country	US
Kind code	B2
Filing date	May 29, 2014
Priority date	May 29, 2013
Publication date	Jan 23, 2018
Grant date	Jan 23, 2018

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.

First claim

What is claimed is:

1. A method for fragmenting a genome comprising:
(a) combining a sample containing genomic DNA with a plurality of Cas9-gRNA complexes, wherein each of the Cas9-gRNA complexes comprises a Cas9 protein and a Cas9-associated guide RNA from a set of at least 10 Cas9-associated guide RNAs that are complimentary to different, pre-defined, sites in the genome, wherein each said different, predefined, site in the genome is followed by a protospacer adjacent motif;
(b) incubating the reaction mixture so that the Cas9 protein cleaves the genomic DNA to produce at least 5 fragments of the genomic DNA having a size within 20% of a chosen size, wherein the chosen size is a length in the range of 1 kb to 100 kb; and
(c) isolating the at least 5 fragments of the genomic DNA from the remainder of the genome.

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 chosen size is in the range of 1 kb to 10 kb.

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

6. The method of claim 5 , wherein the vector is a fosmid, bac or cosmid vector.

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

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

9. 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.

10. 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.

11. The method of claim 10 , wherein the repetitive sequences are cleaved into fragments that range in size from 20 bases to 1 kb.

12. The method of claim 1 , wherein each Cas9-associated guide RNA is a genomic sequence from a different pathogen.

13. The method of claim 1 , wherein said Cas9-associated guide RNAs are in solution.

14. The method of claim 1 , wherein said Cas9-associated guide RNAs are tethered to a substrate in an array.

15. The method of claim 1 , further comprising producing a resultant sample in which the at least 5 fragments of the genomic DNA are enriched to produce a sample which is at least 50% free of other parts of the genome.

16. The method of claim 1 , wherein the at least 5 fragments are from more than one specific region of the genome.

Assignees

• Agilent Technologies Inc

Inventors

• Zeiner Gusti
• Lindstrom Derek Lee
• Peter Brian Jon
• Ach Robert A

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title