The Asymmetric Interaction of Cas9 with Target DNA can Promote High Efficiency Homology‐Directed Genome Editing

Abstract

Cas9 is an RNA‐guided endonuclease that has been adapted from its natural role in CRISPR‐mediated bacterial immunity to enable many applications demanding site‐specific DNA recognition. Wild type Cas9 has been rapidly embraced for genome engineering, either generating knockout cells and organisms via error‐prone non‐homologous end joining (NHEJ) or introducing precise edits via homology directed repair (HDR), while catalytically dead Cas9 (dCas9) fused to various effectors has been used for transcriptional regulation and genomic imaging. However, while Cas9's interaction with target DNA is central to its biological function and use as a genome targeting tool, the molecular nature of this complex is still poorly understood. We have found that the in vitro dissociation of wild type (wt) and dCas9 from double stranded DNA (dsDNA) are both remarkably slow, with a half‐life greater than six hours. However, this stable association is highly asymmetric, and wtCas9 releases only the PAM‐distal end of the non‐target DNA strand post‐cleavage while maintaining contact with the other three strands. dCas9 introduces a bubble into the target DNA that releases the non‐target strand and leaves it accessible for annealing by a complementary single stranded DNA (ssDNA). These activities are present during genome targeting in human cells and can be used to rationally design ssDNA templates that mediate highly efficient gene editing via annealing to the released strand. Optimized templates yield strand‐specific HDR rates over 50% and can furthermore mediate HDR from a single Cas9 nickase or dCas9. Our results highlight Cas9's unusually asymmetric interaction with target DNA, which could be functionally important for CRISPR spacer acquisition or viral interference. Our work also suggests that guiding the formation of specific DNA repair intermediates can dramatically increase the efficiency of genome editing. Because dCas9 is presumably incapable of introducing off‐target breaks in a genome, its use to stimulate HDR may be valuable for precision gene editing in certain therapeutic contexts.