Polynucleotide enrichment using crispr-cas system

Patent metadata

Field	Value
Publication number	US-2025340914-A1
Application number	US-202519268197-A
Country	US
Kind code	A1
Filing date	Jul 14, 2025
Priority date	Jul 21, 2014
Publication date	Nov 6, 2025
Grant date	—

Abstract

A method for enriching a target nucleic acid comprising providing an endonuclease system having a crRNA or a derivative thereof, and a Cas protein or a variant thereof. The crRNA or the derivative thereof contains a target-specific nucleotide region substantially complementary to a region of the target nucleic acid; contacting the target nucleic acid with the endonuclease system to form a complex; and separating the complex and thereby enriching for the target nucleic acid.

First claim

What is claimed:

1 . A method for enriching a plurality of target sequences from a target nucleic acid comprising:
providing a plurality of populations of Cas9 proteins, each population of Cas9 proteins being programmed with a different set of crRNAs, wherein each set of crRNAs contains crRNAs complementary to a different series of regions across the target nucleic acid, contacting the target nucleic acid with each of the plurality of populations of Cas9 proteins in a separate reaction to generate a different series of nucleic acid fragments comprising the target sequences,
wherein at least one of the following is labeled with a binding tag: Cas9 enzyme, crRNA, tracrRNA, and DNA probe targeting the displacement loop, and
separating nucleic acid fragments associated with the binding tags from other fragments, thereby enriching the fragments comprising the target sequences.

2 . The method of claim 1 , wherein the crRNA, sgRNA or tracrRNA comprises a transposon end.

3 . The method of claim 2 , wherein the transposon end is a mosaic end (ME).

4 . The method of claim 2 , further comprising adding a transposase.

5 . The method of claim 4 , wherein the transposase is a Tn5 transposase.

6 . The method of claim 1 , wherein the target nucleic acid is a double-stranded DNA (dsDNA).

7 . The method of claim 1 , wherein the binding tag is biotin.

8 . The method of claim 1 , wherein a Cas9 protein of the plurality of Cas9 proteins retains two nuclease domains and is able to produce a double-stranded DNA break.

9 . The method of claim 1 , wherein a Cas9 protein of the plurality of Cas9 proteins comprises one inactivated nuclease domain comprising a mutation in the domain that cleaves a target nucleic acid strand that is complementary to the crRNA.

10 . The method of claim 9 , wherein said mutation is D10A.

11 . The method of claim 1 , wherein a Cas9 protein of the plurality of Cas9 proteins comprises one inactivated nuclease domain comprising a mutation in the domain that cleaves a target nucleic acid strand that is non-complementary to the crRNA.

12 . The method of claim 11 , wherein said mutation is H840A.

13 . The method of claim 1 , wherein a Cas9 protein of the plurality of Cas9 proteins comprises two inactivated nuclease domains.

14 . The method of claim 13 , wherein the two inactivated nuclease domains comprise a first mutation in the domain that cleaves the strand complementary to the crRNA and a second mutation in the domain that cleaves the strand non-complementary to the crRNA.

15 . The method of claim 14 , wherein said first mutation is D10A and said second mutation is H840A.

Assignees

• Illumina Inc

Inventors

• Cann Gordon M
• Mandell Jeffrey G
• Aravanis Alex
• Norberg Steven
• Pokholok Dmitry K
• Steemers Frank J
• Absalan Farnaz
• Bazargan Leila

Classifications

• C12Q1/6806

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

• C12Q2521/301

  Endonuclease · CPC title

• C12Q1/683

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title