title: In vivo genome editing using Staphylococcus aureus Cas9.
abstract: 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.
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Society of Fellows, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: F Ann
authors.initials: FA
authors.lastname: Ran
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Le
authors.initials: L
authors.lastname: Cong
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Winston X
authors.initials: WX
authors.lastname: Yan
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Scott
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Gootenberg
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Kriz
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.
authors.forename: Bernd
authors.initials: B
authors.lastname: Zetsche
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.
authors.forename: Ophir
authors.initials: O
authors.lastname: Shalem
authors.affiliation: 1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [2] Computational and Systems Biology Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Xuebing
authors.initials: X
authors.lastname: Wu
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA.
authors.forename: Kira S
authors.initials: KS
authors.lastname: Makarova
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA.
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Koonin
authors.affiliation: 1] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [2] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Sharp
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3] Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [4] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000097:blood / Q000378:metabolism; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics*; D008099:Liver / Q000378:metabolism / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000096:biosynthesis / Q000097:blood / Q000172:deficiency / Q000235:genetics; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000097:blood / Q000172:deficiency / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D064130:CRISPR-Associated Proteins; D002784:Cholesterol; C472126:Pcsk9 protein, mouse; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
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