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US 2024/0417787 A1Published application
Polynucleotide amplification using crispr-cas systems
AssigneeIllumina, Inc.
InventorJeffrey G. Mandell
Life of this patent
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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.
Claims22
(canceled)
(canceled)
A method for amplifying a target double-stranded nucleic acid comprising a first strand and a second strand, the method comprising:
- (a) providing a first system comprising:
- a first clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and a first CRISPR-associated (Cas) protein, wherein the first crRNA comprises a target-specific nucleotide region complementary to a region of the first strand;
- (b) 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;
- (c) contacting the target double-stranded nucleic acid with the first system and the second system;
- (d) hybridizing a first primer to the second strand, and hybridizing a second primer to the first strand, wherein one or more of the first primer and second primer is immobilized on a substrate;
- (e) extending a 3′ end of the hybridized first primer and a 3′ end of the hybridized second primer with a polymerase to generate a first and a second double-stranded target nucleic acid;
- and
- (f) repeating step (a) to step (e).
6 more elements
A method for preparing a library of nucleic acids, comprising:
- (a) tagmenting a double-stranded target nucleic acid to obtain a plurality of double-stranded fragments, wherein a first strand of each double-stranded fragment contains a universal sequence;
- and
- (b) amplifying the plurality of double-stranded fragments, comprising:
- (i) 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 the universal sequence;
- (ii) contacting the plurality of double-stranded fragments with the system to form a plurality of complexes, wherein each complex comprises a displaced region of a second strand of a double-stranded fragment of the plurality of double-stranded fragments;
- (iii) hybridizing a primer to the displaced region, wherein the primer comprises a sequence complementary to the displaced region;
- and
- (iv) extending the hybridized primer with a polymerase.
6 more elements
Dependencies are inferred from the claim text.