A Novel Set of Cas9 Fusion Proteins to Stimulate Homologous Recombination: Cas9-HRs

CRISPR- Cas9 has revolutionized genetic engineering. However, the inability to control double-strand break (DSB) repair has severely limited both therapeutic and academic applications. Many attempts have been made to control DSB repair choice. However, particularly in the case of larger edits, none have been able to bypass the rate-limiting step of homologous recombination (HR): long-range 5′ end resection. Here, we describe a novel set of Cas9 fusions, Cas9-HRs, designed to bypass the rate-limiting step of HR repair by simultaneously coupling initial and long-range end resection. Here, we demonstrate that Cas9-HRs can increase the rate of homology directed repair (HDR) by 2- to 2.5-fold and decrease p53 mediated cellular toxicity by two- to fourfold compared to Cas9 and are functional in multiple mammalian cell lines with minimal apparent editing site bias. These properties should make Cas9-HRs an attractive option for applications demanding increased HDR rates for long inserts and/or reduced p53 pathway activation.