Polynucleotide enrichment using crispr-cas system

Abstract

A method for enriching a target nucleic acid comprising providing an endonuclease 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; contacting the target nucleic acid with the endonuclease system to form a complex; and separating the complex and thereby enriching for the target nucleic acid.

1 - 28 . (canceled) 
     
     
         29 . A method for enriching a target double-stranded nucleic acid comprising:
 providing an endonuclease 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;
 contacting the target double-stranded nucleic acid with the endonuclease system to form a first complex; 
 hybridizing a labelled nucleic acid to a second strand of the target double-stranded nucleic acid to form a second complex, the second strand of the target double-stranded nucleic acid being non-complementary to the crRNA or the derivative thereof, and 
 separating the second complex through the labelled nucleic acid, thereby enriching for the target nucleic acid. 
 
     
     
         30 . The method of  claim 29 , further comprising separating the target nucleic acid from the complex. 
     
     
         31 . The method of  claim 30 , further comprising amplifying the targeted nucleic acid. 
     
     
         32 . The method of  claim 29 , wherein the endonuclease system further comprises a trans-activating crRNA (tracrRNA) or a derivative thereof. 
     
     
         33 . The method of  claim 29 , wherein the crRNA or the derivative thereof is a polynucleotide comprising a crRNA polynucleotide fused to a tracrRNA polynucleotide. 
     
     
         34 . The method of  claim 29 , wherein the endonuclease system is a Type II CRISPR-Cas system or a derivative thereof; or
 (v) wherein the target nucleic acid is a double-stranded DNA (dsDNA); or   (vi) wherein, the crRNA is labelled with biotin, and the method optionally further comprises adding streptavidin and thereby separating the complex.   
     
     
         35 . The method of  claim 29 , wherein the Cas protein or the variant thereof is a Cas9 protein or a variant thereof, 
     
     
         36 . The method of  claim 35 , wherein the Cas9 protein or the variant thereof retains two nuclease domains and is able to produce a double-stranded DNA break. 
     
     
         37 . The method of  claim 35 , wherein the Cas9 protein or the variant thereof contains one inactivated nuclease domain comprising a mutation in the domain that cleaves a target nucleic acid strand that is complementary to the crRNA. 
     
     
         38 . The method of  claim 35 , wherein the Cas9 protein or the variant thereof contains two inactivated nuclease domains. 
     
     
         39 . The method of claim  19 , further comprising tagmenting the target nucleic acid. 
     
     
         40 . The method of  claim 29 , further comprising adding a transposase, wherein the crRNA or the derivative thereof contains a transposon end. 
     
     
         41 . The method of  claim 29 , further comprising:
 adding a transposon end to the target nucleic acid, and   tagmenting the target nucleic acid,   wherein the endonuclease system further comprises a transposase   
     
     
         42 . The method of  claim 29 , wherein the target nucleic acid is obtained from a population of cell free DNA (cfDNA) from a subject's plasma or serum, the population of cell free DNA containing the target nucleic acid. 
     
     
         43 . The method of  claim 42 , wherein the subject is a cancer patient. 
     
     
         44 . The method of  claim 29 , wherein the target nucleic acid is in a fetal cell fraction of the cell free DNA, and wherein the cell free DNA is from maternal plasma. 
     
     
         45 . A method for labelling a target nucleic acid comprising:
 providing a first nuclease 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 first target-specific nucleotide region complementary to a first region of the target nucleic acid, and 
       wherein the first Cas protein contains one inactivated nuclease domain;
 contacting a double-stranded nucleic acid containing the target nucleic acid with the first nuclease system to generate a first single-stranded nick at the first region of the target nucleic acid, and 
 labelling the target nucleic acid to facilitate separation.