The clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas) have been widely used for genome editing. CRISPR-Cas system allows for efficient DNA modification when guided by a complementary RNA and in the presence of a protospacer adjacent motif (PAM). However, imperfect guide RNA-target DNA matching may also induce nuclease activity of Cas proteins, resulting in modifications at genomic loci other than the intended locus. This off-target activity could confound research results and constrain clinical utility. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) can be packaged in the payload-limited adeno-associated viral (AAV) vector that is commonly used for in vivo gene editing. Nevertheless, there is still a lack of SaCas9 variants conferring high genome-wide specificity.

Here, rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency were reported. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on GUIDE-seq and targeted deep sequencing analyses. SaCas9-HF can be delivered by AAV and show higher genome-wide specificity than wild-type SaCas9. 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.