Method for fragmenting genomic DNA using CAS9

Patent metadata

Field	Value
Publication number	US-10577644-B2
Application number	US-201615339510-A
Country	US
Kind code	B2
Filing date	Oct 31, 2016
Priority date	May 29, 2013
Publication date	Mar 3, 2020
Grant date	Mar 3, 2020

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 sample containing genomic DNA comprising:
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 one or more genomes, wherein each said different, pre-defined, site in the genome is followed by a protospacer adjacent motif, to produce a reaction mixture;
incubating the reaction mixture to produce at least 5 fragments of genomic DNA, wherein at least some of the fragments are of a length in the range of 1 kb to 200 kb; and
subjecting the fragments to target enrichment.

2. The method of claim 1 , where the different, pre-defined, sites are chosen so that at least 95% of the fragments have a size that is within 20% of a chosen size.

3. The method of claim 2 , where the chosen size is in the range of 1 kb to 10 kb.

4. The method of claim 2 , where the chosen size is in the range of 10 kb to 100 kb.

5. The method of claim 1 , wherein the enrichment comprises:
hybridizing one or more of the fragments to one or more biotinylated probes, wherein each probe hybridizes to both ends of one of the fragments to form a circular fragment;
retrieving the hybridized fragments with magnetic streptavidin beads;
closing the circular fragments by ligation; and
amplifying the circular fragments.

6. The method of claim 1 , wherein the enrichment comprises:
hybridizing one or more of the fragments to one or more biotinylated RNA baits;
retrieving the hybridized fragments with magnetic streptavidin beads; and
amplifying the fragments.

7. The method of claim 1 , wherein one or more of the guide RNAs comprise modified nucleotides.

8. The method of claim 1 , wherein the fragments are subjected to gel electrophoresis.

9. The method of claim 1 , further comprising ligating an adaptor to one or more of the fragments.

10. The method of claim 9 , where the adaptor is a hairpin adaptor.

11. The method of claim 9 , where the adaptor is composed of two distinct oligonucleotide molecules that are base paired with one another.

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

13. The method of claim 12 , further comprising sequencing the fragments without amplification.

14. The method of claim 12 , wherein the fragments are sequenced using nanopore sequencing.

15. The method of claim 12 , wherein the fragments are sequenced using a single molecule sequencing by synthesis method.

16. The method of claim 1 , further comprising insertion of sequences in the genomic DNA at or proximal to said different, predefined sites in one or more genomes.

17. The method of claim 1 , further comprising amplifying one or more of the fragments.

18. The method of claim 1 , wherein the genomic DNA comprises microbial DNA.

19. The method of claim 6 , further comprising:
digesting the RNA baits hybridized to the fragments.

20. The method of claim 1 , where the enrichment comprises:
hybridizing one or more fragments to one or more biotinylated oligonucleotides;
retrieving the hybridized fragments with magnetic streptavidin beads;
and amplifying the fragments.

Assignees

• Agilent Technologies Inc

Inventors

• Peter Brian Jon
• Ach Robert A
• Walter Michael
• Herman Bram

Classifications

• C12Q1/6806 Primary

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

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/1034

  Isolating an individual clone by screening libraries · CPC title

• Y10T436/143333

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