Methods for identifying a target site of a Cas9 nuclease
AssigneePresident and Fellows of Harvard College
InventorsDavid R. Liu, Vikram Pattanayak
Life of this patent
Estimating the term…
Abstract
Some aspects of this disclosure provide strategies, methods, and reagents for determining nuclease target site preferences and specificity of site-specific endonucleases. Some methods provided herein utilize a novel “one-cut” strategy for screening a library of concatemers comprising repeat units of candidate nuclease target sites and constant insert regions to identify library members that can been cut by a nuclease of interest via sequencing of an intact target site adjacent and identical to a cut target site.
Claims24
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.
Dependencies are inferred from the claim text.