The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific. The physical size of the commonly used Cas9 from Streptococcus pyogenes poses challenges for CRISPR-Cas genome editing systems that use the adeno-associated virus as a delivery vehicle; here, smaller Cas9 orthologues are characterized, and Cas9 from Staphylococcus aureus allowed targeting of the cholesterol regulatory gene Pcsk9 in the mouse liver. The RNA-guided endonuclease CRISPR-Cas9 is being widely adopted as the genome-editing tool of choice. However, the physical size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) poses problems for applications that use the adeno-associated virus (AAV) as a delivery vehicle. Feng Zhang and colleagues now characterize six smaller Cas9 orthologues. Focusing on Cas9 from Staphylococcus aureus, they packaged it and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Greater than 40% gene modification was observed within a week of injection, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels.

In vivo genome editing using Staphylococcus aureus Cas9