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. A method of sequencing a target sequence of a target double-stranded nucleic acid comprising
(a) contacting the target double-stranded nucleic acid with a guide RNA sequence having a portion complementary to the target sequence of the target double-stranded nucleic acid and a Cas9 protein,
wherein the guide RNA and the Cas9 protein co-localize to the target sequence of the target double-stranded nucleic acid to form a complex, and
wherein the Cas9 protein is a Cas9 nickase which nicks a strand of the target double-stranded nucleic acid and wherein primer extension is initiated from the nick to incorporate nucleotides to form a growing chain, with the complementary strand serving as a template; and
(b) detecting the nucleotides incorporated into the growing chain, thereby sequencing the target sequence of the target double-stranded nucleic acid.
2. The method of claim 1 wherein the sequencing comprises detecting the incorporation of individual nucleotides into the growing chain.
3. The method of claim 2 where the nucleotides are fluorescently labeled.
4. The method of claim 2 wherein the nucleotides A, C, G, T are differentially labeled and are provided in solution during the incorporation of the nucleotides into the growing chain.
5. The method according to claim 2 wherein the nucleotides are reversible terminators and stepwise sequencing by synthesis is conducted.
6. The method of claim 2 where the nucleotides are labeled at a terminal phosphate and real-time sequencing is conducted.
7. The method of claim 1 wherein the target double-stranded nucleic acid is analyzed as a linear string.
8. The method of claim 1 wherein the target double-stranded nucleic acid is analyzed as a linear string that is substantially stretched out.
9. The method of claim 1 wherein primer extension from the nick is initiated from multiple sites on a target double-stranded nucleic acid linear string.
10. The method of claim 1 wherein a plurality of target sequences of a plurality of target double-stranded nucleic acids are sequenced in parallel.
11. The method of claim 1 wherein the target double-stranded nucleic acid is a genomic DNA molecule, and wherein one or more regions of the genomic DNA molecule are sequenced.
12. The method of claim 1 wherein the gRNA/Cas9 complex is removed after nicking.
13. The method of claim 1 wherein the target double-stranded nucleic acid is analysed in a cell in situ.
14. The method of claim 13 wherein the cell is fixed before analysis.
15. The method of claim 13 wherein RNA molecules within the cell are removed before analysis.
16. The method of claim 1 wherein the Cas9 nickase comprises an inactive RuvC-like domain.
17. The method of claim 1 wherein the Cas9 nickase comprises an inactive HNH domain.
18. The method of claim 1 wherein the Cas9 nickase is a D10A nicking mutant.
19. The method of claim 1 wherein the Cas9 nickase is an H840A nicking mutant.