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

RNA-guided Cas9 endonuclease based genome editing (CRISPR-Cas9) is widely applied in biomedical sciences. However, unwanted off-target activities could confound research results and constrain its clinical utilities. Although there are Streptococcus pyogenes Cas9 (SpCas9) variants with minimal off-target activities, no high-fidelity variants are available for Staphylococcus aures Cas9 (SaCas9), which is much smaller and thus more versatile than the most commonly used SpCas9. We hypothesize that reducing the affinity of those SaCas9 sites which are in close contact with sgRNA/DNA target heteroduplex can destabilize the interaction between SaCas9 and mismatched heteroduplex (off-target sites) without compromising its on-target activity. We performed structure-guided protein engineering to introduce one or more engineered sites to those interacting SaCas9 sites. We evaluated on-target activities of 6 SaCas9 variants by using enhanced green fluorescent protein (EGFP) disruption assay and targeted deep sequencing. Genome-wide off-target activities of these SaCas9 variants in human cell were assessed by using GUIDE-seq. We aim to improve SaCas9 specificity for genome-editing where high specificity is needed. Our data showed that variant 4M demonstrated the least off-target sites, expanded PAM and highest on/off target reads ratio than wild type SaCas9, suggesting the highest specificity. In general, this research provides an alternative to wild-type SaCas9 for genome-editing applications requiring exceptional specificity.