Vitro isolation and enrichment of nucleic acids using site-specific nucleases

Abstract

The present invention relates to a method for the isolation of a target nucleic acid region. In particular, said method comprises the steps of contacting a population of nucleic acid molecules with at least one Type II Cas protein-gRNA complex, wherein said gRNA comprises a guide segment that is complementary to the sequence comprised in the target region of at least one nucleic acid molecule, thereby forming a Type II Cas protein-gRNA-nucleic acid complex, contacting the population of nucleic acid molecules with at least one enzyme having exonuclease activity, and isolating the target nucleic acid region from the Type II Cas protein-gRNA-nucleic acid complex.

1 .- 15  (canceled) 
     
     
         16 . Method of isolating a target region of a nucleic acid molecule comprising the steps of:
 a. contacting a population of nucleic acid molecules with at least one Type II Cas protein-gRNA complex, wherein the gRNA comprises a guide segment that is complementary to a sequence comprised in the target region of at least one nucleic acid molecule, thereby forming a Type II Cas protein-gRNA-nucleic acid complex,   b. contacting the population of nucleic acid molecules with at least one enzyme having exonuclease activity, thus degrading nucleic acid molecules that are not comprised in the Type II Cas protein-gRNA-nucleic acid complex.   
     
     
         17 . The method of  claim 16 , wherein the Type II Cas protein is Cas9 or Cpf1. 
     
     
         18 . The method of  claim 17 , wherein the Type II Cas protein is a nickase or is catalytically inactive. 
     
     
         19 . The method of  claim 16 , wherein the enzyme having exonuclease activity is a lambda exonuclease and/or exonuclease I. 
     
     
         20 . The method of  claim 16 , further comprising contacting the population of nucleic acid molecules with at least one protector molecule prior to, simultaneously, or after step a), wherein the protector molecule is a hairpin adaptor or a site-specific endonuclease. 
     
     
         21 . The method of  claim 20 , wherein the site-specific endonuclease is selected from the group consisting of: a TALEN, a zinc-finger protein, and at least one additional Type II Cas protein-gRNA complex. 
     
     
         22 . The method of  claim 16 , further comprising fragmentation of the population of nucleic acid molecules. 
     
     
         23 . The method of  claim 21 , wherein the fragmentation is performed by contacting the population of nucleic acid molecules with at least one site-specific endonuclease. 
     
     
         24 . The method of  claim 16 , wherein the target region has a length of at least 44 nucleotides. 
     
     
         25 . The method of  claim 16 , wherein the population of nucleic acid molecules comprises one or more cfDNA molecules. 
     
     
         26 . The method of  claim 16 , wherein the target region comprises one or more repeat regions, rearrangements, duplications, translocations, deletions, mismatches, SNPs, or modified bases. 
     
     
         27 . The method of  claim 16 , wherein the ratio of target nucleic acid:Type II Cas protein:gRNA is at least 1:10:10. 
     
     
         28 . The method of  claim 16 , further comprising step c) of isolating the target region from the Type II Cas protein-gRNA-nucleic acid complex formed in step a). 
     
     
         29 . The method of  claim 28 , further comprising step d) wherein at least one single-stranded nucleic acid molecule is hybridized to a single-stranded region of the target molecule of step c) 
     
     
         30 . The method of  claim 29 , wherein the at least one single-stranded nucleic acid molecule is hybridized to a 3′ overhang of the target molecule of step c). 
     
     
         31 . The method of  claim 29 , wherein the single-stranded nucleic acid molecule hybridizes at least 50 nucleotides from the Type II Cas protein protospacer adjacent motif. 
     
     
         32 . The method of  claim 28 , wherein the isolated nucleic acid is used as a substrate for detecting protein binding to nucleic acid. 
     
     
         33 . The method of  claim 16 , wherein prior to isolation the nucleic acid target region comprises less than 10% of the total initial nucleic acid in a sample. 
     
     
         34 . The method of  claim 16 , wherein at least two target regions are isolated. 
     
     
         35 . The method of  claim 26 , wherein the modified bases are epigenetic modifications.