Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

ABSTRACT: The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot

SUBMITTER: Wolfs JM 

PROVIDER: S-EPMC5206545 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature