Compositions and Methods of Nucleic Acid-Targeting Nucleic Acids

Abstract

This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof. Genome engineering can refer to altering the genome by deleting, inserting, mutating, or substituting specific nucleic acid sequences. The altering can be gene or location specific. Genome engineering can use nucleases to cut a nucleic acid thereby generating a site for the alteration. Engineering of non-genomic nucleic acid is also contemplated.

1 - 26 . (canceled) 
     
     
         27 . A method for enrichment and sequencing of a target nucleic acid having a 5′ end and a 3′ end, comprising:
 contacting a first complex and a second complex with a polynucleotide comprising the target nucleic acid,
 the first complex comprising a catalytically active Cas9 protein and a first nucleic acid-targeting nucleic acid comprising a spacer that hybridizes to a first site in the polynucleotide, wherein the first site flanks the 5′ end of the target nucleic acid, and the first complex binds and cleaves the first site, and 
 the second complex comprising a catalytically active Cas9 protein and a second nucleic acid-targeting nucleic acid comprising a spacer that hybridizes to a second site in the polynucleotide, wherein the second site flanks the 3′ end of the target nucleic acid, and the second complex binds and cleaves the second site, 
 wherein cleavage of the first site and the second site produces an excised target nucleic acid; 
 
 purifying the excised target nucleic acid; and 
 sequencing the excised target nucleic acid. 
 
     
     
         28 . The method of  claim 27 , wherein the catalytically active Cas9 protein of the first complex is a  Streptococcus pyogenes  Cas9 protein. 
     
     
         29 . The method of  claim 27 , wherein the catalytically active Cas9 protein of the second complex is a  Streptococcus pyogenes  Cas9 protein. 
     
     
         30 . The method of  claim 27 , wherein each of the catalytically active Cas9 protein of the first complex and the catalytically active Cas9 protein of the second complex is a  Streptococcus pyogenes  Cas9 protein. 
     
     
         31 . The method of  claim 27 , wherein the first nucleic acid-targeting nucleic acid comprises RNA. 
     
     
         32 . The method of  claim 27 , wherein the second nucleic acid-targeting nucleic acid comprises RNA. 
     
     
         33 . The method of  claim 27 , wherein the first nucleic acid-targeting nucleic acid comprises DNA. 
     
     
         34 . The method of  claim 27 , wherein the second nucleic acid-targeting nucleic acid comprises DNA. 
     
     
         35 . The method of  claim 27 , wherein the first nucleic acid-targeting nucleic acid is a single guide nucleic acid-targeting nucleic acid. 
     
     
         36 . The method of  claim 27 , wherein the second nucleic acid-targeting nucleic acid is a single guide nucleic acid-targeting nucleic acid. 
     
     
         37 . The method of  claim 27 , wherein each of the first nucleic acid-targeting nucleic acid and the second nucleic acid-targeting nucleic acid is a single guide nucleic acid-targeting nucleic acid. 
     
     
         38 . The method of  claim 27 , wherein the polynucleotide comprises DNA. 
     
     
         39 . The method of  claim 38 , wherein the DNA is genomic DNA. 
     
     
         40 . The method of  claim 39 , wherein the genomic DNA is eukaryotic genomic DNA. 
     
     
         41 . The method of  claim 39 , wherein the genomic DNA is derived from lysed cells. 
     
     
         42 . The method of  claim 39 , wherein the genomic DNA is purified genomic DNA. 
     
     
         43 . The method of  claim 27 , wherein purifying the excised target nucleic acid further comprises ligating adapters to the 5′ end and the 3′ end of the excised target nucleic acid. 
     
     
         44 . The method of  claim 27 , wherein purifying the excised target nucleic acid further comprises amplification of the excised target nucleic acid. 
     
     
         45 . The method of  claim 27 , wherein the excised target nucleic acid is purified by one or more method selected from the group consisting of gel electrophoresis, size-selective elution, and precipitation. 
     
     
         46 . The method of  claim 27 , wherein the sequencing of the excised target nucleic acid is by one or more method selected from the group consisting of pyrosequencing, sequencing by ligation, sequencing by synthesis using modified nucleotides, sequencing by ion detection technologies, nanopore-based sequencing, capillary sequencing, electronic sequencing, single molecule sequencing, droplet microfluidic sequencing, sequencing by hybridization, and bisulfate sequencing.