Patent ID: 9163284
Filing Date: 2015-10-20
Classification: A61K,A61P,C12N,C12Q,C12Y

Abstract:
1. A method for identifying a target site of an RNA-programmable nuclease, the method comprising (a) providing an RNA-programmable nuclease that cuts a double-stranded nucleic acid target site, wherein cutting of the target site results in cut nucleic acid strands comprising a 5′ phosphate moiety; (b) contacting the RNA-programmable nuclease of (a) with a library of candidate nucleic acid molecules, wherein each nucleic acid molecule comprises a concatemer of a sequence comprising a candidate nuclease target site and a constant insert sequence of at least 10 and not more than 70 nucleotides, under conditions suitable for the RNA-programmable nuclease to cut a candidate nucleic acid molecule comprising a target site of the RNA-programmable nuclease; and (c) identifying nuclease target sites cut by the RNA-programmable nuclease in (b) by direct sequencing of an intact nuclease target site on the nucleic acid strand that was cut by the RNA-programmable nuclease in step (b), wherein the method does not include computational reconstruction of nuclease target sites from cut half-sites.