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.
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All reagents described in this manuscript have been deposited with Addgene (plasmid IDs 61591, 61592 and 61593). Source data are available online and deep sequencing data are available at Sequence Read Archive under BioProject accession number PRJNA274149.
We thank E. Charpentier, I. Fonfara and K. Chylinski for discussions; A. Scherer-Hoock, B. Clear and the MIT Division of Comparative Medicine for assistance with animal experiments; Boston Children’s Hospital Viral Core and R. Xiao for assistance with AAV production; N. Crosetto for advice on BLESS; C.-Y. Lin and I. Slaymaker for experimental assistance; and the entire Zhang laboratory for support and advice. F.A.R. is a Junior Fellow at the Harvard Society of Fellows. W.X.Y. is supported by T32GM007753 from the National Institute of General Medical Sciences and a Paul and Daisy Soros Fellowship. J.S.G. is supported by a US Department of Energy Computational Science Graduate Fellowship. X.W. is a Howard Hughes Medical Institute International Student Research Fellow. P.A.S. is supported by United States Public Health Service grants RO1-GM34277, R01-CA133404 from the National Institutes of Health, and PO1-CA42063 from the National Cancer Institute, and partially by Cancer Center Support (core) grant P30-CA14051 from the National Cancer Institute. F.Z. is supported by the National Institutes of Health through NIMH (5DP1-MH100706) and NIDDK (5R01DK097768-03), a Waterman Award from the National Science Foundation, the Keck, New York Stem Cell, Damon Runyon, Searle Scholars, Merkin, and Vallee Foundations, and B. Metcalfe. F.Z. is a New York Stem Cell Foundation Robertson Investigator. The Children’s Hospital virus core is supported by an NIH core grant (5P30EY012196-17). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. CRISPR reagents are available to the academic community through Addgene, and information about the protocols, plasmids, and reagents can be found at the Zhang laboratory website http://www.genome-engineering.org.
Extended data figures
This file contains a Supplementary Discussion, Supplementary References, Supplementary Tables 1-9 and Supplementary Sequences.