Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared to SpCas9 in Genome Editing

A series of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) systems have been engineered for genome editing. The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus. However, a comparison of their detailed gene editing outcomes is still lacking. By characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells (iPSCs) and K562 cells, we found that SaCas9 could edit the genome with greater efficiency than SpCas9. We also compared the spacer lengths of single-guide RNA (sgRNA, 18–21 nt for SpCas9 and 19–23 nt for SaCas9) and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9, respectively. However, the optimal spacer size for a particular guide RNA ranged from 18–21 nt or 21–22 nt for SpCas9 and SaCas9, respectively. Furthermore, SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining (NHEJ) +1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif (PAM), characteristic of a staggered cut. Accordingly, editing with SaCas9 led to higher knock-in efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertion or adeno-associated virus serotype 6 (AAV6) donor-mediated homology-directed repair (HDR). Finally, GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9. Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.