Polynucleotide enrichment and amplification using CRISPR-Cas or Argonaute systems

Patent metadata

Field	Value
Publication number	US-12391983-B2
Application number	US-202218063865-A
Country	US
Kind code	B2
Filing date	Dec 9, 2022
Priority date	May 11, 2016
Publication date	Aug 19, 2025
Grant date	Aug 19, 2025

Abstract

A method for enriching or amplifying a target nucleic acid including providing a system having a guide nucleic acid, and a Cas or Argonaute protein or a variant thereof. The guide nucleic acid 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 preparing a library of nucleic acids, comprising:
(i) 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 (ii) amplifying the plurality of double-stranded fragments, comprising: (a) providing a system comprising: a 5′ phosphorylated single-stranded nucleic acid, and an Argonaute protein, wherein the 5′ phosphorylated single-stranded nucleic acid comprises a sequence complementary to a region of the universal sequence,
(b) contacting the plurality of double-stranded fragments with the system to form a plurality of complexes, wherein each complex comprises the 5′ phosphorylated single-stranded nucleic acid hybridized to the first strand, and a displaced region of a second strand of a double-stranded fragment of the plurality of double-stranded fragments, wherein the displaced region comprises a loop,
(c) hybridizing a primer to the displaced region, wherein the primer comprises a sequence complementary to the displaced region, and
(d) extending the hybridized primer with a polymerase.

2. The method of claim 1 , wherein the tagmenting comprises contacting the target nucleic acid with a transposome comprising a transposase and a nucleic acid comprising the universal sequence.

3. The method of claim 1 , wherein the amplification is linear.

4. The method of claim 1 , wherein the amplification is exponential.

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

6. The method of claim 1 , wherein the target nucleic acid is a double-stranded DNA or a double-stranded RNA.

7. The method of claim 1 , wherein the 5′ phosphorylated single-stranded nucleic acid is a single-stranded DNA having a length of 25 nucleotides or less.

8. The method of claim 2 , wherein the transposase is selected from a hyperactive Tn5 transposase or a MuA transposase.

9. The method of claim 1 , further comprising (e) sequencing the extended primer.

10. The method of claim 1 , wherein the system is bound to a surface.

11. The method of claim 10 , wherein a plurality of beads comprises the surface.

12. The method of claim 10 , wherein a flow cell comprises the surface.

13. The method of claim 3 , wherein the amplification is performed under isothermal conditions.

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

15. The method of claim 1 , wherein the Argonaute protein is Natronobacterium gregorgi Argonaute.

16. The method of claim 1 , wherein the universal sequence comprises a nucleotide sequence selected from any one of SEQ ID NOs: 01-06.

17. The method of claim 1 , wherein the double-stranded target nucleic acid comprises genomic DNA.

18. A method for preparing a library of nucleic acids, comprising:
(i) 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, wherein the double-stranded target nucleic acid is a double-stranded RNA; and
(ii) amplifying the plurality of double-stranded fragments, comprising:
(a) providing a system comprising: a 5′ phosphorylated single-stranded nucleic acid, and an Argonaute protein, wherein the 5′ phosphorylated single-stranded nucleic acid comprises a sequence complementary to a region of the universal sequence,
(b) contacting the plurality of double-stranded fragments with the system to form a plurality of complexes, wherein each complex forms a loop comprising the 5′ phosphorylated single-stranded nucleic acid hybridized to the first strand, and a displaced region of a second strand of a double-stranded fragment of the plurality of double-stranded fragments,
(c) hybridizing a primer to the displaced region, wherein the primer comprises a sequence complementary to the displaced region, and
(d) extending the hybridized primer with a polymerase.

Assignees

• Illumina Inc

Inventors

• Mandell Jeffrey G
• He Molly

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q2531/101

  Linear amplification, i.e. non exponential · CPC title

• C12Q2522/101

  Single or double stranded nucleic acid binding proteins · CPC title