Polynucleotide amplification using CRISPR-Cas systems
First Claim
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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), anda 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.
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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.
59 Citations
24 Claims
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1. A method for amplifying a target double-stranded nucleic acid comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for amplifying a target double-stranded nucleic acid comprising:
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(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. - View Dependent Claims (22, 23, 24)
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Specification