Polynucleotide amplification using CRISPR-Cas systems

Patent metadata

Field	Value
Publication number	US-12065695-B2
Application number	US-202016735372-A
Country	US
Kind code	B2
Filing date	Jan 6, 2020
Priority date	Nov 11, 2014
Publication date	Aug 20, 2024
Grant date	Aug 20, 2024

Abstract

A method for amplifying a target nucleic acid including providing a 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, and contacting the target nucleic acid with the system to form a complex.

First claim

What is claimed is:

1. A method for amplifying a target double-stranded nucleic acid comprising a first strand and a second strand, comprising:
(a) providing a system comprising: a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and a CRISPR-associated (Cas) protein, wherein the crRNA contains a target-specific nucleotide region complementary to a region of the first strand;
(b) contacting the target double-stranded nucleic acid with the system to form a complex comprising a displaced region of the second strand;
(c) hybridizing a primer to the displaced region, wherein the primer is immobilized on a substrate, and wherein the primer comprises a sequence complementary to the displaced region, and
(d) extending a nucleic acid complementary to the second strand from the primer using a polymerase.

2. The method of claim 1 , wherein the substrate comprises a flow cell.

3. The method of claim 1 , wherein the substrate comprises a bead.

4. The method of claim 1 , further comprising repeating step (a) to step (d).

5. The method of claim 4 , wherein the target double-stranded nucleic acid is exponentially amplified.

6. The method of claim 1 , wherein the system is selected from the group consisting of a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, and a Type III CRISPR-Cas system.

7. The method of claim 1 , wherein the first strand of the target double-stranded nucleic acid comprises a sequence complementary to a 5′-NGG protospacer-adjacent motif (PAM).

8. The method of claim 1 , wherein the first strand of the target double-stranded nucleic acid comprises a universal sequence, and the primer comprises a sequence of a region of the universal sequence.

9. The method of claim 8 , wherein the universal sequence comprises the sequence of SEQ ID NO:03 or SEQ ID NO:04.

10. The method of claim 1 , wherein the primer comprises the sequence of SEQ ID NO:05 or SEQ ID NO:06.

11. The method of claim 1 , wherein the polymerase is a strand-displacing polymerase selected from the group consisting of Bst, Bsu, and Phi29.

12. The method of claim 1 , further comprising:
providing a second system comprising: a second crRNA and a second Cas protein, wherein the second crRNA comprises a target-specific nucleotide region complementary to a region of the second strand;
contacting the target double-stranded nucleic acid with the second system;
hybridizing a second primer to the first strand contacted with the second system, and
extending the hybridized second primer.

13. The method of claim 1 , wherein the system further comprises a trans-activating crRNA (tracrRNA).

14. The method of claim 13 , wherein the crRNA is fused to the tracrRNA.

15. The method of claim 1 , wherein the crRNA comprises the sequence of SEQ ID NO: 07 or 08.

16. The method of claim 1 , wherein the target double-stranded nucleic acid comprises the sequence of SEQ ID NO:09 or 10.

17. The method of claim 1 , wherein the primer comprises the sequence of SEQ ID NO:12 or 14.

Assignees

• Illumina Inc

Inventors

• Mandell Jeffrey G

Classifications

• C12Q2522/101

  Single or double stranded nucleic acid binding proteins · CPC title

• C12Q2521/101

  DNA polymerase · CPC title

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title