RNA-Guided Systems For Probing And Mapping Of Nucleic Acids
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)