In vivo genome editing using Staphylococcus aureus Cas9

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.National Institute of General Medical Sciences (U.S.) (T32GM007753)Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.)National Institutes of Health (U.S.) (United States Public Health Service Grant RO1-GM34277)National Institutes of Health (U.S.) (United States Public Health Service Grant R01-CA133404)National Cancer Institute (U.S.) (PO1-CA42063)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institute of Mental Health (U.S.) (5DP1-MH100706)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (5R01DK097768-03)National Science Foundation (U.S.) (Waterman Award)W. M. Keck FoundationNew York Stem Cell FoundationDamon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramMerkin FoundationVallee Foundatio