Researchers developed a smaller and more efficient Cas9 enzyme from Staphylococcus aureus (SaCas9) for in vivo genome editing. SaCas9 is approximately 1 kb shorter than the commonly used Streptococcus pyogenes (SpCas9) and exhibits similar genome editing efficiency. The study demonstrated that SaCas9 can be packaged into a single adeno-associated virus (AAV) vector along with its single-guide RNA (sgRNA) expression cassette, enabling efficient and specific genome editing in mice. When targeting the cholesterol regulatory gene Pcsk9 in the mouse liver, SaCas9 achieved over 40% gene modification within one week, resulting in significant reductions in serum Pcsk9 and total cholesterol levels. The study also showed that SaCas9 has high specificity for genome editing in vivo, as confirmed by unbiased profiling using BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). This method identified candidate off-target cleavage sites, and no significant indels were observed in liver tissue. The SaCas9 system was found to be well-tolerated, with no signs of acute toxicity observed up to four weeks post-injection. The findings suggest that SaCas9 has the potential for highly efficient, specific, and well-tolerated in vivo genome editing. The study highlights the importance of understanding the rules governing Cas9 nuclease activity to improve the predictive power of computational guide design programs. The AAV-SaCas9 system is a promising tool for therapeutic applications, particularly in targeting genes involved in cholesterol homeostasis.Researchers developed a smaller and more efficient Cas9 enzyme from Staphylococcus aureus (SaCas9) for in vivo genome editing. SaCas9 is approximately 1 kb shorter than the commonly used Streptococcus pyogenes (SpCas9) and exhibits similar genome editing efficiency. The study demonstrated that SaCas9 can be packaged into a single adeno-associated virus (AAV) vector along with its single-guide RNA (sgRNA) expression cassette, enabling efficient and specific genome editing in mice. When targeting the cholesterol regulatory gene Pcsk9 in the mouse liver, SaCas9 achieved over 40% gene modification within one week, resulting in significant reductions in serum Pcsk9 and total cholesterol levels. The study also showed that SaCas9 has high specificity for genome editing in vivo, as confirmed by unbiased profiling using BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). This method identified candidate off-target cleavage sites, and no significant indels were observed in liver tissue. The SaCas9 system was found to be well-tolerated, with no signs of acute toxicity observed up to four weeks post-injection. The findings suggest that SaCas9 has the potential for highly efficient, specific, and well-tolerated in vivo genome editing. The study highlights the importance of understanding the rules governing Cas9 nuclease activity to improve the predictive power of computational guide design programs. The AAV-SaCas9 system is a promising tool for therapeutic applications, particularly in targeting genes involved in cholesterol homeostasis.