Identification of Guide-Intrinsic Determinants of Cas9 Specificity.

ABSTRACT: Considerable effort has been devoted to developing a comprehensive understanding of CRISPR nuclease specificity. In silico predictions and multiple genome-wide cellular and biochemical approaches have revealed a basic understanding of the Cas9 specificity profile. However, none of these approaches has delivered a model that allows accurate prediction of a CRISPR nuclease's ability to cleave a site based entirely on the sequence of the guide RNA (gRNA) and the target. We describe a library-based biochemical assay that directly reports the cleavage efficiency of a particular Cas9-guide complex by measuring both uncleaved and cleaved target molecules over a wide range of mismatched library members. We applied our assay using libraries of targets to evaluate the specificity of Staphy

SUBMITTER: Huston NC 

PROVIDER: S-EPMC6694761 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature