Polynucleotide amplification using CRISPR-Cas systems

• US 10,577,649 B2
• Filed: 11/10/2015
• Issued: 03/03/2020
• Est. Priority Date: 11/11/2014
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Mandell, Jeffrey G.
• Primary Examiner(s): Gonzalez, Antonio Galisteo
• Application Number:
  US15/524,962
• Publication Number:
  US 20170342474A1
• Time in Patent Office: 1,575 Days
• CPC Class Codes: C12N 15/10 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 Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site

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.

Claims

1. A method for amplifying a target double-stranded nucleic acid comprising:

(a) providing a system having;

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 a first strand of the target double-stranded nucleic acid;

(b) contacting the target double-stranded nucleic acid with the system to form a complex;

(c) hybridizing a primer to a second strand of the target double-stranded nucleic acid, the primer containing a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and

(d) extending a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer using a polymerase.

2. The method of claim 1, further comprising repeating step (a) to step (d) for one or more times.

3. The method of claim 1, wherein the target double-stranded nucleic acid is linearly amplified.

4. The method of claim 1, wherein the target double-stranded nucleic acid is exponentially amplified.

5. 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.

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

7. The method of claim 1, wherein the crRNA is a polynucleotide comprising a crRNA polynucleotide fused to a trans-activating crRNA (tracrRNA) polynucleotide.

8. The method of claim 1, wherein the first strand of the target double-stranded nucleic acid contains a sequence complementary to a 5′

-NGG protospacer-adjacent motif (PAM).

9. The method of claim 1, wherein the first strand of the target double-stranded nucleic acid contains a universal sequence, and wherein the crRNA contains a sequence complementary to a region of the universal sequence.

10. The method of claim 9, wherein the primer contains a sequence of a region of the universal sequence.

11. The method of claim 9, wherein the universal sequence has a sequence of SEQ ID No. 3 or SEQ ID No. 4.

12. The method of claim 11, wherein the crRNA contains a sequence of SEQ ID No. 7 or SEQ ID No. 8.

13. The method of claim 12, wherein the primer contains a sequence of SEQ ID No. 5 or SEQ ID No. 6.

14. The method of claim 1, wherein the Cas protein is selected from the group consisting of a Cas9 protein, a Cascade protein, and a Cas3 protein.

15. The method of claim 14, wherein the Cas9 protein contains two inactivated nuclease domains, comprising 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, wherein the first mutation is D10A and the second mutation is H840A.

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

17. The method of claim 1, further comprising:

applying at least one transposase and at least one transposon end composition containing a transferred strand to a sample containing a target nucleic acid under conditions where the target nucleic acid and the transposon end composition undergo a transposition reaction to generate a mixture, wherein the target nucleic acid is fragmented to generate a plurality of target nucleic acid fragments, and

incorporating a universal primer sequence into each of the plurality of target nucleic acid fragments,

wherein the crRNA contains a target-specific nucleotide region complementary to a region of the universal primer.

18. The method of claim 17, wherein the universal primer is incorporated into the plurality of target nucleic acid fragments by a polymerase chain reaction.

19. The method of claim 18, wherein the universal primer has sequence complementary to a 5′

-NGG protospacer-adjacent motif (PAM).

20. The method of claim 17, wherein two universal primers are incorporated into two ends of each of the plurality of target nucleic acid fragments.

21. A method for amplifying a target double-stranded nucleic acid comprising:

(a) providing a first system having;

a first clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) or a first trans-activating crRNA (tracrRNA), and

a first CRISPR-associated (Cas) protein,

wherein the first crRNA or the first tracrRNA contains a target-specific nucleotide region complementary to a region of a first strand of the target double-stranded nucleic acid;

(b) providing second system having;

a second crRNA or a second tracrRNA, and

a second Cas protein,

wherein the second crRNA or the second tracrRNA contains a target-specific nucleotide region complementary to a region of a second strand of the target double-stranded nucleic acid;

(c) contacting the target double-stranded nucleic acid with the first system and the second system;

(d) hybridizing a first primer to a second strand of the target double-stranded nucleic acid, the first primer containing a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and hybridizing a second primer to a first strand of the target double-stranded nucleic acid, the second primer containing a sequence complementary to a region of the first strand of the target double-stranded nucleic acid, and

(e) extending the 3′

end of the first primer and the second primer with one or more polymerases to generate a first and a second double stranded target nucleic acid.

22. The method of claim 21, wherein:

the first strand of the target double-stranded nucleic acid contains a first universal sequence, and wherein the crRNA of the first system contains a sequence complementary to a region of the first universal sequence, and

the second strand of the target double-stranded nucleic acid contains a second universal sequence, and wherein the crRNA of the second system contains a sequence complementary to a region of the second universal sequence.

23. The method of claim 22, wherein the first primer contains a sequence of a region of the first universal sequence, and the second primer contains a sequence of a region of the second universal sequence.

24. The method of claim 23, wherein:

the first universal sequence has a sequence of SEQ ID No. 3, the crRNA of the first system contains a sequence of SEQ ID No. 7, and the first primer contains a sequence of SEQ ID No. 5, and

the second universal sequence has a sequence of SEQ ID No. 4, the crRNA of the second system contains a sequence of SEQ ID No. 8, and the second primer contains a sequence of SEQ ID No. 6.