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.
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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.
Patent applications have been filed as to the subject matter of the manuscript such as, for example, US patent 8,865,406 issued 21 October 2014 and US patent 8,895,308 issued 25 November 2014. F.Z. is a founder of Editas Medicine and scientific advisor for Editas Medicine and Horizon Discovery.
Extended data figures and tables
a, Distribution of lengths for Cas9 > 600 Cas9 orthologues19. b, Schematic of Type II CRISPR-Cas loci and sgRNA from eight bacterial species. Spacer or ‘guide’ sequences are shown in blue, followed by direct repeats (grey). Predicted tracrRNAs are shown in red, and folded based on the Constraint Generation RNA folding model50.
Stacked bar graph indicates the fraction of targets cleaved at 2, 3, 4, or 5 bp upstream of PAM for each Cas9 orthologue; most Cas9 enzymes cleave stereotypically at 3 bp upstream of PAM (red triangle).
a, SURVEYOR assays showing indel formation at human endogenous loci from co-transfection of Cas9 orthologues and sgRNA. PAM sequences for individual targets are shown above each lane, with the consensus region for each PAM highlighted in red. Red triangles indicate cleaved fragments. b, SaCas9 generates indels efficiently for a multiple targets. c, Box-whisker plot of indel formation as a function of SaCas9 guide length L, with unaltered guides (perfect match of L nucleotides, grey bars) or replacement of the 5′-most base of guide with guanine (G + L − 1 nucleotides, blue bars) (n = 8 guides).
a, Schematic of the Staphylococcus aureus subspecies aureus CRISPR locus. b, Schematic of SaCas9 sgRNA with 21-nucleotide guide, crRNA repeat (grey), tetraloop (black) and tracrRNA (red). The number of crRNA repeat to tracrRNA anti-repeat base-pairing is indicated above the grey boxes. SaCas9 cleaves targets with varying repeat:anti-repeat lengths in c, HEK 293FT and d, Hepa1-6 cell lines. (n = 3, error bars show s.e.m.)
a, Unbiased identification of PAM motif for dSaCas9 and dSpCas9. Peaks were analysed for the best match by motif score to the guide region only within 50 nucleotides of the peak summit. The alignment was extended for 10 nucleotides at the 3′ end and visualized using Weblogo. Numbers in parentheses indicate the number of called peaks. b, Histograms show the distribution of the peak summit relative to motif for dSaCas9 and dSpCas9. Position 1 on x axis indicates the first base of PAM.
Indels at top off-target sites predicted by dCas9-ChIP for each Cas9 and sgRNA pair, based on ChIP peaks ranked by sequence similarity of the genomic loci to the guide motif (heat map in purple), or P value of ChIP enrichment over control (heat map in red). Lines connect the common targets (EMX1) and off-targets between the two Cas9 enzymes.
a, Overview of the data analysis pipeline starting from the raw sequencing reads. Representative sequencing read mappings and corresponding histograms of the pairwise distances between all the forward orientation (red) reads and reverse orientation (blue) reads, displayed for representative b, DSB hotspots and poorly defined DSB sites and c, Cas9-induced DSBs with detectable indels. Fraction of pairwise distances between reads overlapping by no more than 6 bp (dashed vertical line) are indicated over histogram plots.
List of top off-target sites ranked by DSB scores for each Cas9 and sgRNA pair. Indel levels are determined by targeted deep sequencing. Blue triangles indicate positions of peak BLESS signal, and where present, PAMs and targets with sequence homology to the guide are highlighted. Lines connect the common on-targets (EMX1) and off-targets between the two Cas9 enzymes. N.D., not determined.
Extended Data Figure 9 Indel measurements of top candidate off-target sites based on sequence similarity score.
Off-targets are predicted based on sequence similarity to on-target, accounting for number and position of Watson–Crick base-pairing mismatches as previously described22. NNGRR and NRG are used as potential PAMs for SaCas9 and SpCas9, respectively. Lines connect the common targets (EMX1) and off-targets between the two Cas9 enzymes. Correlation plots between indel percentages and b, prediction based on sequence similarity, c, ChIP peaks ranked by motif similarity, or d, DSB scores for top ranking off-target loci. Trendlines, r2, and P values are calculated using ordinary least squares.
a, Schematics illustrating the mouse Apob gene locus and the positions of the three guides tested. b, Experimental time course and c, SURVEYOR assay showing indel formation at target loci after intravenous injection of AAV2/8 carrying thyroxine-binding globulin (TBG) promoter-driven SaCas9 and U6-driven guide at 2 × 1011 total genome copies (n = 1 animal each). d, Oil-red staining of liver tissue from AAV- or saline-injected animals. Male C56BL/6 mice were injected at 8 weeks of age and analysed 4 weeks post injection.
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Ran, F., Cong, L., Yan, W. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). https://doi.org/10.1038/nature14299
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