POLYNUCLEOTIDE AMPLIFICATION USING CRISPR-CAS SYSTEMS

• US 20170342474A1
• Filed: 11/10/2015
• Published: 11/30/2017
• Est. Priority Date: 11/11/2014
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Mandell, Jeffrey G.
• Granted Patent: US 10,577,649 B2
• Time in Patent Office: 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) or a derivative thereof, and

a CRISPR-associated (Cas) protein or a variant thereof,

wherein the crRNA or the derivative thereof 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 target nucleic acid is a double-stranded DNA (dsDNA).

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

7. The method of claim 1, wherein the system is a Type I CRISPR-Cas system or a derivative thereof.

8. The method of claim 1, wherein the system is a Type II CRISPR-Cas system or a derivative thereof.

9. The method of claim 1, wherein the system is a Type III CRISPR-Cas system or a derivative thereof.

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

11. The method of claim 1, wherein the crRNA or the derivative thereof is a polynucleotide comprising a crRNA polynucleotide fused to a tracrRNA polynucleotide.

12. 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).

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

14. The method of claim 13, wherein the primer contains a sequence of a region of the universal sequence.

15. The method of claim 13, wherein the universal sequence has a sequence of SEQ ID No. 3.

16. The method of claim 15, wherein the crRNA contains a sequence of SEQ ID No.7.

17. The method of claim 16, wherein the primer contains a sequence of SEQ ID No. 5.

18. The method of claim 13, wherein the universal sequence has a sequence of SEQ ID No. 4.

19. The method of claim 18, wherein the crRNA contains a sequence of SEQ ID No.8.

20. The method of claim 19, wherein the primer contains a sequence of SEQ ID No. 6.

21. The method of claim 1, wherein the Cas protein or the variant thereof is a Cas9 protein or a variant thereof.

22. The method of claim 21, wherein the Cas9 protein contains two inactivated nuclease domains.

23. The method of claim 22, 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.

24. The method of claim 23, wherein the first mutation is D10A and the second mutation is H840A.

25. The method of claim 1, wherein the Cas protein or the variant thereof is a Cascade protein or a variant thereof.

26. The method of claim 1, wherein the Cas protein or the variant thereof is a Cas3 protein or a variant thereof.

27. The method of claim 1, wherein the polymerase is a strand-displacing polymerase.

28. The method of claim 27, wherein the polymerase is selected from a group consisting of Bst, Bsu, and Phi29.

29. 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 or the derivative thereof contains a target-specific nucleotide region complementary to a region of the universal primer.

30. The method of claim 29, wherein the universal primer is incorporated into the plurality of target nucleic acid fragments by a PCR reaction.

31. The method of claim 30, wherein the universal primer has sequence complementary to a 5′

-NGG protospacer-adjacent motif (PAM).

32. The method of claim 30, wherein the universal sequence has a sequence of SEQ ID No. 3.

33. The method of claim 32, wherein the crRNA contains a sequence of SEQ ID No.7.

34. The method of claim 33, wherein the primer contains a sequence of SEQ ID No.5.

35. The method of claim 30, wherein the universal primer has a sequence of SEQ ID No. 4.

36. The method of claim 35, wherein the crRNA contains a sequence of SEQ ID No.8.

37. The method of claim 36, wherein the primer contains a sequence of SEQ ID No. 6.

38. The method of claim 29, wherein two universal primers are incorporated into two ends of each of the plurality of target nucleic acid fragments.

39. The method of claim 38, wherein the two universal primers have sequences of SEQ ID No.3 and SEQ ID No.4.

61. The method of anyone of claims 1-60, wherein the target nucleic acid is genomic DNA.

62. The method of anyone of claims 1-60, wherein the target nucleic acid contains chromosomal DNA or a fragment thereof.

63. The method of anyone of claims 1-60, wherein the target nucleic acid comprises a genome or a partial genome.

64. The method of anyone of claims 1-63, further comprising sequencing the target nucleic acid or target nucleic acid fragments.

65. The method of claim 64, wherein the sequencing comprises use of one or more of sequencing by synthesis, bridge PCR, chain termination sequencing, sequencing by hybridization, nanopore sequencing, and sequencing by ligation.

40. 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 derivative thereof, and

a first CRISPR-associated (Cas) protein or a variant thereof,

wherein the first crRNA or the derivative thereof 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 clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) or a derivative thereof, and

a second CRISPR-associated (Cas) protein or a variant thereof,

wherein the second crRNA or the derivative thereof 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.

41. The method of claim 40, further comprising repeating step (a) to step (e) for one or more times.

42. The method of claim 40, wherein the target nucleic acid is a double-stranded DNA (dsDNA).

43. The method of claim 40, wherein the target nucleic acid is a double-stranded RNA (dsRNA).

44. The method of claim 40, wherein the first system or the second system is a Type I CRISPR-Cas system or a derivative thereof.

45. The method of claim 40, wherein the first system or the second system is a Type II CRISPR-Cas system or a derivative thereof.

46. The method of claim 40, wherein the first system or a second system is a Type III CRISPR-Cas system or a derivative thereof.

47. The method of claim 40, wherein the first system or the second system further comprises a trans-activating crRNA (tracrRNA) or a derivative thereof.

48. The method of claim 40, wherein the crRNA or the derivative thereof of the first system or the second system is a polynucleotide comprising a crRNA polynucleotide fused to a tracrRNA polynucleotide.

49. The method of claim 40, wherein the first strand and the second strand of the target double-stranded nucleic acid contain a sequence complementary to a 5′

-NGG protospacer-adjacent motif (PAM).

50. The method of claim 40, wherein:

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

51. The method of claim 50, 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.

52. The method of claim 51, wherein:

the first universal sequence has a sequence of SEQ ID No. 3, the crRNA or the derivative thereof 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 or derivative thereof of the second system contains a sequence of SEQ ID No.8, and the second primer contains a sequence of SEQ ID No. 6.

53. The method of claim 40, wherein the Cas protein or the variant thereof of the first system or the second system is a Cas9 protein or a variant thereof.

54. The method of claim 53, wherein the Cas9 protein contains two inactivated nuclease domains.

55. The method of claim 54, 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.

56. The method of claim 55, wherein the first mutation is D10A and the second mutation is H840A.

57. The method of claim 40, wherein the Cas protein or the variant thereof of the first system or the second system is a Cascade protein or a variant thereof.

58. The method of claim 40, wherein the Cas protein or the variant thereof of the first system or the second system is a Cas3 protein or a variant thereof.

59. The method of claim 40, wherein the polymerase is a strand-displacing polymerase.

60. The method of claim 59, wherein the polymerase is selected from a group consisting of Bst, Bsu, and Phi29.