Nucleic acid enrichment using cas9

Abstract

A method of enriching for a fragment of a genome, as well as corresponding compositions and kits, are provided. In certain embodiments, the method comprises: (a) contacting a sample comprising fragmented DNA with a Cas9-gRNA complex comprising mutant Cas9 protein that has inactivated nuclease activity and a Cas9-associated guide RNA that is complementary to a site in the DNA, to produce a Cas9-fragment complex that comprises a fragment of the fragmented DNA; and (b) isolating the complex. In addition, other methods and compositions for Cas9/CRISPR-mediated nucleic acid manipulation are also provided.

What is claimed is: 
     
         1 . A method for labeling a site in a nucleic acid, comprising:
 (a) contacting the nucleic acid with a labeled complex comprising a mutant Cas9 protein that has inactivated nuclease activity and a guide RNA under conditions by which the complex binds to the nucleic acid; and   (b) analyzing the product of step (a).   
     
     
         2 . The method of  claim 1 , wherein the guide RNA is fluorescently labeled. 
     
     
         3 . The method of  claim 1 , wherein the Cas9 protein is fluorescently labeled. 
     
     
         4 . The method of  claim 1 , wherein the complex is bound by a fluorescently labeled antibody. 
     
     
         5 . The method of  claim 1 , wherein the complex is labeled with streptavidin protein. 
     
     
         6 . The method of  claim 1 , wherein the product of step (a) is analyzed by fluorescence microscopy. 
     
     
         7 . The method of  claim 6 , wherein the fluorescence microscopy is coupled with microfluidic or nanofluidic analysis. 
     
     
         8 . The method of  claim 1 , wherein the labeled Cas9-gRNA complex binds to a test chromosome comprising the nucleic acid, and a signal from the test chromosome is compared with a signal of a reference chromosome. 
     
     
         9 . The method of  claim 8 , wherein the reference chromosome is from a healthy or wild-type organism. 
     
     
         10 . The method of  claim 1 , wherein the nucleic acid is in a test chromosome, and a plurality of labeled Cas9-gRNA complexes contacts the test chromosome, and the same contacts a reference chromosome, and signals from the test chromosome are compared to signals from the reference chromosome. 
     
     
         11 . A method comprising contacting a sample comprising fragmented genomic DNA with a Cas9-gRNA complex comprising a mutant Cas9 protein that has inactivated nuclease activity and a Cas9-associated guide RNA that comprises a modified nucleotide and is complementary to a site in said genomic DNA, to produce a Cas9-fragment complex that comprises a fragment of the fragmented genomic DNA. 
     
     
         12 . The method of  claim 11 , wherein the modified nucleotide comprises a fluorescent label. 
     
     
         13 . The method of  claim 11 , wherein the modified nucleotide comprises a hapten. 
     
     
         14 . The method of  claim 11 , wherein the modified nucleotide comprises a methylated purine or pyrimidine, an acylated purine or pyrimidine, an alkylated ribose or other heterocycle. 
     
     
         15 . The method of  claim 11 , wherein the modified nucleotide comprises a modification on a sugar moiety. 
     
     
         16 . The method of  claim 15 , wherein the sugar moiety is modified by one or more hydroxyl groups being replaced with halogen atoms or aliphatic groups or functionalized as ethers or amines. 
     
     
         17 . The method of  claim 11 , wherein the modified nucleotide comprises a thioethyl group at a 2′ position of the nucleotide. 
     
     
         18 . The method of  claim 11 , wherein the modified nucleotide comprises a modified base selected from 4-thiouridine, 5-bromouridine, 5-iodouridine, and 6-thioguanosine. 
     
     
         19 . A composition comprising a mutant Cas9 protein that has inactivated nuclease activity and a plurality of Cas9-associated guide RNA that are complementary to different sites in genomic DNA, wherein the Cas9-associated guide RNA comprises a modified nucleotide. 
     
     
         20 . The composition of  claim 19 , wherein the modified nucleotide comprises biotin.