Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity.

RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on GUIDE-seq and targeted deep sequencing analyses. Among fifteen tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at nine sites, minimal off-target activities at six sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among four known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild-type. When delivered by an adeno associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild-type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to -off target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.