RNA-Guided Systems For Probing And Mapping Of Nucleic Acids

Abstract

Methods of detecting, probing, mapping and directed sequencing of target nucleic acids are provided using a guide RNA and a Cas9 protein. Methods for detecting the binding of the guide RNA/Cas9 complex to a target nucleic acid where the guide RNA includes a 3′ tail sequence that can hybridize to a probe are provided. Methods for detecting the binding of the guide RNA/Cas9 complex to a target nucleic acid where the complex is physically detected are provided.

1 .- 38 . (canceled) 
     
     
         39 . A method of detecting a target nucleic acid sequence comprising
 contacting the target nucleic acid sequence with a guide RNA sequence having a portion complementary to the target nucleic acid sequence and a Cas9 protein,   wherein the guide RNA and the Cas9 protein co-localize to the target nucleic acid sequence to form a complex, and   wherein the complex is detected thereby detecting the target nucleic acid sequence.   
     
     
         40 . The method of  claim 39  wherein the guide RNA includes a detectable label. 
     
     
         41 . The method of  claim 39  wherein the Cas9 protein includes a detectable label. 
     
     
         42 . The method of  claim 39  wherein the complex includes a detectable label. 
     
     
         43 . The method of  claim 39  wherein the complex is detected by a nanopore. 
     
     
         44 . The method of  claim 39  wherein the complex is detected by electron microscopy. 
     
     
         45 . The method of  claim 39  wherein the complex is detected by scanning probe microscopy. 
     
     
         46 . The method of  claim 39  wherein the complex is detected by a cantilever. 
     
     
         47 . The method of  claim 39  wherein the complex is detected by a quartz crystal microbalance. 
     
     
         48 . The method of  claim 39  wherein the complex is detected by a field effect transistor. 
     
     
         49 . The method of  claim 39  wherein the guide RNA includes a 3′ tail sequence complementary to a probe sequence. 
     
     
         50 . The method of  claim 39  wherein the guide RNA includes a 3′ tail sequence complementary to a probe sequence including a detectable label, and the probe sequence is bound to the 3′ tail sequence. 
     
     
         51 . The method of  claim 39  wherein the guide RNA includes a 3′ tail sequence complementary to a probe sequence including a plurality of detectable labels, and the probe sequence is bound to the 3′ tail sequence. 
     
     
         52 . The method of  claim 39  wherein the guide RNA includes a 3′ tail sequence complementary to a probe sequence including a detectable label, and the probe sequence is bound to the 3′ tail sequence, and wherein the probe sequence is amplified. 
     
     
         53 . The method of  claim 39  wherein the guide RNA includes a 3′ tail sequence as a binding pair to a probe or detectable label. 
     
     
         54 . The method of  claim 39  wherein the target nucleic acid is double stranded genomic DNA. 
     
     
         55 . The method of  claim 39  wherein the target nucleic acid is chromosomal DNA. 
     
     
         56 .- 59 . (canceled) 
     
     
         60 . The method of  claim 39  wherein the Cas9 protein is wild type Cas9, a cas9 nickase or a nuclease null Cas9. 
     
     
         61 . The method of  claim 39  wherein a detectable label is directly or indirectly bound to the Cas9 protein. 
     
     
         62 . The method of  claim 39  wherein a detectable label is directly or indirectly bound to the guide RNA. 
     
     
         63 . The method of  claim 39  wherein a detectable label is directly or indirectly bound to the complex. 
     
     
         64 .- 76 . (canceled)