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In vivo genome Staphylococcus aureus Cas9

In vivo genome editing using Staphylococcus aureus Cas9

F.Ann Ran 1,2*,Le Cong 1,3*,Winston X.Yan 1,4,5*,David A.Scott 1,6,7,Jonathan S.Gootenberg 1,8,Andrea J.Kriz 3,Bernd Zetsche 1,Ophir Shalem 1,Xuebing Wu 9,10,Kira S.Makarova 11,Eugene V.Koonin 11,Phillip A.Sharp 3,9&Feng Zhang 1,6,7,12

The RNA-guided endonuclease Cas9has emerged as a versatile genome-editing platform.However,the size of the commonly used Cas9from 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 Cas9orthologues and show that Cas9from Staphylococcus aureus (SaCas9)can edit the genome with efficiencies similar to those of SpCas9,while being more than 1kilobase shorter.We packaged SaCas9and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9in the mouse liver.Within one week of injection,we observed .40%gene modification,accompanied by significant reductions in serum Pcsk9and total cholesterol levels.We further assess the genome-wide targeting specificity of SaCas9and SpCas9using BLESS,and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.Cas9,an RNA-guided endonuclease derived from the type II CRISPR-Cas bacterial adaptive immune system 1–7,has been harnessed for genome editing 8,9and holds tremendous promise for biomedical research.Genome editing of somatic tissue in postnatal animals,however,has been limited in part by the challenge of delivering Cas9in vivo .For this purpose,adeno-associated virus (AAV)vectors are attractive vehicles 10because of their low immunogenic potential,reduced oncogenic risk from host-genome integration 11,and broad range of serotype specificity 12–15.Nevertheless,the restrictive cargo size (,4.5kb,excluding the inverted terminal repeats)of AAV presents an obstacle for packaging the com-monly used Streptococcus pyogenes Cas9(SpCas9,,4.2kb)and its single guide RNA (sgRNA)in a single vector;although technically feasible 17,this approach leaves little room for customized expression and control elements 16.

In search of smaller Cas9enzymes for efficient in vivo delivery by AAV,we have previously described a short Cas9from the CRISPR1locus of Streptococcus thermophilus LMD-9(St1Cas9,,3.3kb)8as well as a rationally-designed truncated form of SpCas9(ref.18)for genome editing in human cells.However,both systems have important prac-tical drawbacks:the former requires a complex protospacer-associated motif (PAM)sequence (NNAGAAW)3,which restricts the range of accessible targets,whereas the latter exhibits reduced activity.Given the substantial diversity of CRISPR-Cas systems present in sequenced microbial genomes 19,we therefore sought to interrogate and discover additional Cas9enzymes that are small,efficient and broadly targeting.

In vitro cleavage by small Cas9enzymes

Type II CRISPR-Cas systems require only two main components for eukaryotic genome editing:a Cas9enzyme,and a chimaeric sgRNA 6derived from the CRISPR RNA (crRNA)and the noncoding trans-activating crRNA (tracrRNA)4,20.Analysis of over 600Cas9orthologues

shows that these enzymes are clustered into two length groups with characteristic protein sizes of approximately 1,350and 1,000amino acid residues,respectively 19,21(Extended Data Fig.1a),with shorter Cas9enzymes having significantly truncated REC domains (Fig.1a).From these shorter Cas9enzymes,which belong to Type IIA and IIC subtypes,we selected six candidates for profiling (Fig.1a and Extended Data Fig.1b).To determine the cognate crRNA and tracrRNA for each Cas9,we computationally identified regularly interspaced repeat sequences (direct repeats)within a 2-kb window flanking the CRISPR locus.We then predicted the tracrRNA by detecting sequences with strong com-plementarity to the direct repeat sequence (an anti-repeat region),at least two predicted stem-loop structures,and a Rho-independent tran-scriptional termination signal up to 150nucleotides downstream of the anti-repeat region.Although a truncated tracrRNA can support robust DNA cleavage in vitro 6,previous reports show that the secondary struc-tures of the tracrRNA are important for Cas9activity in mammalian cells 8,9,18,22.Therefore,we designed sgRNA scaffolds for each ortho-logue by fusing the 39end of a truncated direct repeat with the 59end of the corresponding tracrRNA,including the full-length tail,via a 4-nucleotide linker 6(Extended Data Fig.1b and Supplementary Table 1).To identify the PAM sequence for each Cas9,we first constructed a library of plasmid DNA containing a constant 20-bp target followed by a degenerate 7-bp sequence (59-NNNNNNN).We then incubated cell lysate from human embryonic kidney 293FT (293FT)cells expressing the Cas9orthologue with its in vitro -transcribed sgRNA and the plas-mid library.By generating a consensus from the 7-bp sequence found on successfully cleaved DNA plasmids (Fig.1b),we determined putative PAMs for each Cas9(Fig.1c).We observed that,similar to SpCas9,most Cas9orthologues cleaved targets 3-bp upstream of the PAM (Extended Data Fig.2).To validate each putative PAM from the library,we then incubated a DNA template bearing the consensus PAM with cell lysate

*These authors contributed equally to this work.

Broad Institute of MIT and Harvard,Cambridge,Massachusetts 02142,USA.2Society of Fellows,Harvard University,Cambridge,Massachusetts 02138,USA.3Department of Biology,Massachusetts Institute of Technology,Cambridge,Massachusetts 02139,USA.4Graduate Program in Biophysics,Harvard Medical School,Boston,Massachusetts 02115,USA.5Harvard-MIT Division of Health Sciences and Technology,Harvard Medical School,Boston,Massachusetts 02115,USA.6McGovern Institute for Brain Research,Massachusetts Institute of Technology,Cambridge,Massachusetts 02139,USA.7

Department of Brain and Cognitive Sciences,Massachusetts Institute of Technology,Cambridge,Massachusetts 02139,USA.8Department of Systems Biology,Harvard Medical School,Boston,Massachusetts 02115,USA.9David H.Koch Institute for Integrative Cancer Research,Massachusetts Institute of Technology,Cambridge,Massachusetts 02139,USA.10Computational and Systems Biology Graduate Program,Massachusetts Institute of Technology,Cambridge,Massachusetts 02139,USA.11National Center for Biotechnology Information,National Library of Medicine,National Institutes of Health,Bethesda,Maryland 20894,USA.12Department of Biological Engineering,Massachusetts Institute of Technology,Cambridge,Massachusetts 02139,USA.00M O N T H 2015|V O L 000|N A T U R E |1

and the corresponding sgRNA.We found that the Cas9orthologues,in combination with the sgRNA designs,successfully cleaved the appro-priate targets (Fig.1d and Supplementary Table 2).

To test whether each Cas9orthologue can facilitate genome editing in mammalian cells,we co-transfected 293FT cells with individual Cas9enzymes and their respective sgRNAs targeting human endoge-nous loci containing the appropriate PAMs.Of the six Cas9orthologues tested,only the one from Staphylococcus aureus (SaCas9)produced indels with efficiencies comparable to those of SpCas9(Extended Data Fig.3a,b and Supplementary Table 3),suggesting that DNA-cleavage activity in cell-free assays does not necessarily predict activity in mam-malian cells.These observations prompted us to focus on harnessing SaCas9and its sgRNA for in vivo applications.

SaCas9sgRNA design and PAM discovery

Although mature crRNAs in S.pyogenes are processed to contain 20-nucleotide spacers (guides)and 19-to 22-nucleotide direct repeats 4,RNA sequencing of crRNAs from other organisms reveals that the spacer and direct repeat sequence lengths can vary 4,20,23.We therefore tested sgRNAs for SaCas9with variable guide lengths and repeat:anti-repeat duplexes.We found that SaCas9achieves the highest editing efficiency in mammalian cells with guides between 21and 23nucleotides long

and can accommodate a range of lengths for the direct repeat:anti-repeat region (Fig.2a,b,Extended Data Fig.4).This notably contrasts with SpCas9,where the natural 20-nucleotide guide length can be truncated to 17nucleotides without significantly compromising nuclease acti-vity,while increasing specificity 24.Additionally,replacing the first base of the guide with guanine further improved SaCas9activity (Extended Data Fig.3c).

To fully characterize the SaCas9PAM and the seed region within its guide sequence 25,we performed chromatin immunoprecipitation (ChIP)using catalytically mutant forms of SaCas9(dSaCas9,D10A and N580A mutations,based on homology to SpCas9)or SpCas9(dSpCas9,D10A and H840A mutations)and their corresponding sgRNAs.We targeted two loci in the human EMX1gene with composite NGGRRT PAMs,which allow targeting by both dCas9s.A search for motifs containing both the guide region and PAM within 50nucleotides of the ChIP peak summits revealed seed sequences of 7–8nucleotides for dSaCas9(Fig.2c).In addition,NNGRRT and NGG PAMs were found adjacent to the seed sequences for dSaCas9and dSpCas9,respectively (Extended Data Fig.5).Although the 6th position of the PAM is pre-dominantly thymine,we did observe low levels of degeneracy in both the biochemical and ChIP-based PAM discovery assays (Fig.1c and Extended Data Fig.5a).We therefore tested the base preference for this position and determined that,although SaCas9cleaves genomic targets most efficiently with NNGRRT,all NNGRR PAMs can be cleaved and should be considered as potential targets,especially in the context of off-target evaluations (Fig.2d,Extended Data Fig.6and Supplemen-tary Table 4).

Unbiased profiling of Cas9specificity

As advances in Cas9technology promise to enable a broad range of in vivo and therapeutic applications,accurate,genome-wide identifica-tion of off-target nuclease activity has become increasingly important.Although a number of studies have employed sequence similarity-based off-target search 22,26–30or dCas9-ChIP 31,32to predict off-target sites for Cas9,such approaches cannot assess the nuclease activity of Cas9in a comprehensive and unbiased manner.To measure the

genome-wide

1,3681,0531,1211,1301,0821,0031,0371,084

aa:R u v C

Cas9

R u v C I I H N H

R u v C

I I I

P. lava C. diph S. aure N. cine C. lari S. pyog S. ther 1

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PAM:

T A

C C A T T C

A G C A T G T G G A C A G A G G T T T G

A G G T G A A C A G T G A C G T T T G T A T G G A G T A

A T G A A T A T A G A A T A

T G G G G

C A A T G G G G C A G G A T A T A G G A G G A T C A G A A A T T A G A A A

––

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B i t s B i t s

S. pyogenes S. thermophilus 1

C. diphtheriae

B i t s

P. lavamentivorans

B i t s DNA substrate with randomized PAM

Purify cleaved fragments

Adapter ligation

PAM identification

Cas9 cleavage

Barcode

Barcode Sequencing b

Sequencing PAM position:6543217

5′3′

IIC - Neisseria cinerea ATCC 14685IIC - Campylobacter lari CF89-12

IIC - Corynebacterium diphtheriae NCTC 13129

IIC - Parvibaculum lavamentivorans DS-1IIA - Streptococcus pyogenes SF370IIA - Streptococcus thermophilus LMD-9 CRISPR1

IIA - Streptococcus pasteurianus ATCC 43144

IIA - Staphylococcus aureus subsp. aureus

Figure 1|Biochemical screen for small Cas9orthologues.a ,Phylogenetic tree of selected Cas9orthologues.Subfamily and sizes (amino acids)are

indicated,with nuclease domains highlighted in coloured boxes,and conserved sequences in black.b ,Schematic illustration of the in vitro cleavage-based method used to identify the first seven positions (59-NNNNNNN)of protospacer adjacent motifs (PAMs).c ,Consensus PAMs for eight Cas9

orthologues from sequencing of cleaved fragments.Error bars are Bayesian 95%confidence interval 45.d ,Cleavage using different orthologues and sgRNAs targeting loci bearing the putative PAMs (consensus shown in red).Red triangles indicate cleavage fragments.

3′-5′-5′-c

Guide length (nt)

I n d e l (%, H e p a 1-6)

EMX1-sg1 (7,257 peaks)

NNGRR 89%

NNGRRT 71%EMX1-sg2 (12,964 peaks)

Preference for NNGRR(N )

I n d e l (%, 293F T )

A C G T

(N ) =Seed

PAM

–20–19–18–17–16–15–14–13–12–11–10–9–8–7–6–5–4–3–2–11234567

B i t s

–20–19–18–17–16–15–14–13–12–11–10–9–8–7–6–5–4–3–2–11234567

Seed PAM

B i t s

67%91%

NNGRR NNGRRT Peaks containing motif+PAM:2102

Peaks containing motif+PAM:Figure 2|Characterization of Staphylococcus aureus Cas9(SaCas9)in 293FT cells.a ,SaCas9sgRNA scaffold (red)and guide (blue)base-pairing at target locus (black)immediately 59of PAM.b ,Box-whisker plot showing indel levels vary depending on the length of the guide sequence (n 54).

c ,dSaCas9-ChIP reveals peaks associate

d with seed 1PAM.Text to th

e right indicates the total number o

f peaks and percentage containin

g significant (false discovery rate ,0.1)matc

h to the guide motif followed by NNGRRT or NNGRR PAMs.d ,Pooled indel values for NNGRR(A),(C),(G),or (T)PAM combinations (n 512,21,39and 44,respectively).

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cleavage activity of SaCas9and SpCas9directly,we applied BLESS(direct in situ breaks labelling,enrichment on streptavidin and next-generation sequencing)33to capture a snapshot of Cas9-induced DNA double-stranded breaks(DSBs)in cells.We transfected293FT cells with SaCas9or SpCas9and the same EMX1targeting guides used in the previous ChIP experiment,or pUC19as a negative control.After cells are fixed,free genomic DNA ends from DSBs are captured using biotinylated adaptors and analysed by deep sequencing(Fig.3a).To identify candidate Cas9-induced DSB sites genome-wide,we estab-lished a three-step analysis pipeline following alignment of the sequenced BLESS reads to the genome(Extended Data Fig.7a,Supplementary Discussion).First,we applied nearest-neighbour clustering on the aligned reads to identify groups of DSBs(DSB clusters)across the genome.Second,we sought to separate potential Cas9-induced DSB clusters from background DSB clusters resulting from low frequency biological processes and technical artefacts,as well as high-frequency telomeric and centromeric

33From the on-target and a

small subset of verified off-target sites(predicted by sequence similarity

using a previously established method22and sequenced to detect indels),

we found that reads in Cas9-induced

DSB clusters mapped to charac-

teristic,well-defined genomic positions compared

to the more diffuse

alignment pattern at background DSB clusters.To distinguish between

the two types of DSB clusters,we calculated in each cluster the distance

between all possible pairs of forward and reverse-oriented reads(cor-

responding to39and59ends of DSBs),and filtered out the background

DSB clusters based on the distinctive pairwise-distance distribution of

these clusters(Extended Data Fig.7b,c).Third,the DSB score for a given

locus was calculated

by comparing the count of DSBs in the experi-

mental and negative control samples using a maximum-likelihood

estimate22(Supplementary Discussion).This analysis identified the

on-target loci for both SaCas9and SpCas9guides as the top scoring

sites,and revealed additional sites with high DSB scores(Fig.3b–d).

Next,we sought

to assess whether DSB scores correlated with indel

formation.We used targeted deep sequencing to detect indel

formation

Fix cells

Shear and

capture

Label

distal end

Enrich and

sequence

Deproteinize

nuclei

DSB

proximal-labelling

Indel (%, 293FT)Indel (%, 293FT)

EMX1-sg1EMX1-sg2

S r

Target

SaCas9SpCas9

0.1

0.01

Sequence similarity score

DSB score

(

)

(

)

SaCas9SpCas9

Figure3|Characterization of genome-wide nuclease activity of SaCas9and

SpCas9.a,Schematic of BLESS processing steps.b,Manhattan plots of

genome-wide DSB clusters generated by each Cas9and sgRNA pair,with

on-target loci shown above(see Supplementary Discussion).c,Correlation

between DSB scores and indel levels for top-scoring DSB clusters.Trend lines,

r2and P values are calculated using ordinary least squares method.d,Off-target

loci from BLESS with detectable indels through targeted deep sequencing

(n53)are shown.Heat maps indicate DSB score(blue),motif score from ChIP

(purple),or sequence similarity score(green)for each locus.Blue triangles

indicate peak positions of BLESS signal.

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on the,30top-ranking off-target sites identified by BLESS for each Cas9and sgRNA combination.We found that only those sites that contained PAM and homology to the guide sequence exhibited indels (Extended Data Fig.8).We observed a strong linear correlation between DSB scores and indel levels for each Cas9and sgRNA pairing(r250.948 and0.989for the two EMX1targets with SaCas9and r250.941and 0.753for those with SpCas9)(Fig.3c,Extended Data Fig.9b–d).Fur-thermore,BLESS identified additional off-target sites not previously predicted by sequence similarity to target or ChIP(Extended Data Figs7 and9,Supplementary Tables5and6).These new off-target sites include not only those containing Watson–Crick base-pairing mismatches to the guide,but also the recently reported insertion and deletion mis-matches in the guide:target heteroduplex(Fig.3d)29,30.Together,these results highlight the need for more precise understanding of rules governing Cas9nuclease activity,a requisite step towards improving the predictive power of computational guide design programs.

In vivo genome editing using SaCas9

Following in vitro characterization,we incorporated SaCas9and its sgRNA into an AAV vector to test its efficacy and specificity in vivo. The small size of SaCas9enables packaging of both a U6-driven sgRNA and a cytomegalovirus(CMV)-or thyroxine-binding globulin(TBG)-driven SaCas9expression cassette into a single AAV vector within the4.5-kb packaging https://www.wendangku.net/doc/ea1164142.html,ing hepatocyte-tropic AAV serotype8,we targeted the mouse apolipoprotein (Apob)gene(Extended Data Fig.10a).One week after intravenous administration of virus into C57BL/6mice,we observed,5%indel formation in liver tissue;after four weeks,the liver tissue showed characteristic hepatic lipid accumulation from Apob knockdown following histology analysis using oil red staining34–37(Extended Data Fig.10b,c).

We next targeted proprotein convertase subtilisin/kexin type9 (Pcsk9),a therapeutically relevant gene involved in cholesterol home-ostasis38.Inhibitors of the human convertase PCSK9have emerged as a promising new class of cardioprotective drugs,after human genetic studies revealed that loss of PCSK9is associated with a reduced risk of cardiovascular disease and lower levels of low-density lipoprotein (LDL)cholesterol39–41.We designed two Pcsk9-targeting sgRNAs (20-nucleotide guides with additional59guanine)and validated their activity in vitro.Each sgRNA was packaged into AAV-SaCas9and injected into mice(231011total genome copies)(Fig.4a).One week after administration,we observed greater than40%indel formation at either locus in whole liver tissue,with similar levels two and four weeks post-injection(Fig.4b).To determine the effect of Pcsk9-tar-geting AAV-SaCas9dosage on serum Pcsk9and total cholesterol levels,we administered a range of AAV titres from0.531011to 431011total genome copies.With all titres,we observed a,95% decrease in serum Pcsk9and a,40%decrease in total cholesterol one week after administration,both of which were sustained throughout the course of four weeks(Fig.4c,d).

Days post injection

(

–

)

In vitro target

validation

(Week 2)

Virus production

(Week 3)

Tail vein injection

(Weeks 4 — )

Tissue analysis

Indel (%, liver)

Pcsk9-sg1Pcsk9-sg2

Indel (%, liver)

(

)

bGHpA

Thyroxine-binding globulin

Guide

< 4.7 kb

Days post injection

(

)

(

)

Figure4|AAV-delivery of SaCas9for in vivo genome editing.a,Single-

vector AAV system and experimental timeline.b,Indels at Pcsk9targets in liver

tissue following injection of AAV at231011total genome copies(n53

animals).c,d,Time course of serum Pcsk9(c)and total cholesterol in animals

(d;n53for all titres and time points,error bars show s.e.m.).e,Manhattan

plots of BLESS-identified DSB clusters in N2a cells.Inset indicates indel levels

at top DSB scoring loci.f,Indels in liver tissue(n53animals,error bars

indicate Wilson intervals)at BLESS-identified off-target loci from N2a cells.

Heat map indicates DSB scores.

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We next considered SaCas9off-target modifications in the liver tissue samples.To first identify candidate off-target cleavage sites for the two Pcsk9-targeting guides,we transiently transfected an AAV-CMV::SaCas9vector into mouse Neuroblastoma-2a (N2a)cells and applied BLESS to detect Cas9-induced DSBs in the genome.For both guides,we found very low levels of DSB signal across the genome except at the on-target loci (Fig.4e).Targeted deep sequencing of the candidate off-target sites identified by BLESS in N2a cells did not reveal appreciable levels of indels in either N2a cells or liver tissue (4weeks post injection of 231011total genome copies)(Fig.4e,f and Supplementary Table 8).We addi-tionally sequenced off-target sites predicted by target sequence sim-ilarity,and likewise did not detect indel formations (Supplementary Table 9).

Finally,we examined the titre-matched Pcsk9-targeting and enhanced green fluorescent protein-conjugated (EGFP)TBG–EGFP cohorts as well as naive animals for signs of toxicity or acute immune response.At 1week post-injection,necropsy and gross examination of liver tissue of the cohorts revealed no abnormalities;further histological examina-tion of the liver by haematoxylin and eosin (H&E)staining showed no signs of inflammation,such as aggregates of lymphocytes or macro-phages (Fig.5a).Throughout the time course of the experiment,there were no elevated levels of serum alanine aminotransferase (ALT),albu-min,and total bilirubin in any of the cohorts.We observed a slight trend in aspartate transaminase (AST)increase across all cohorts at four weeks,including the uninjected animals.The elevated levels did not exceed the upper limit of normal and is not indicative of hepatocellular injury in animals (Fig.5b).However,a larger cohort study should be conducted to further evaluate the potential side-effects of Cas9-mediated in vivo genome editing.In addition,the differences between mouse and human

immune responses need to be better elucidated before considering this approach for therapeutic applications.

Discussion

Here,we develop a small and efficient Cas9from S.aureus for in vivo genome editing 17.The results of these experiments highlight the power of using comparative genomic analysis 19,42in expanding the CRISPR-Cas9toolbox.Identification of new Cas9orthologues 19,42,in addition to structure-guided engineering,could yield a repertoire of Cas9variants with expanded capabilities and minimized molecular weight,for nuc-leic acid manipulation to further advance genome and epigenome engineering.

The AAV-SaCas9system is able to mediate efficient and rapid edit-ing of Pcsk9in the mouse liver,resulting in reductions of serum Pcsk9and total cholesterol levels.To assess the specificity of SaCas9,we used an unbiased DSB detection method,BLESS,to identify a list of can-didate off-target cleavage sites in a mouse cell line.We examined these sites in liver tissue transduced by AAV-SaCas9and did not observe any indel formation within the detection limits of in vitro BLESS and targeted deep sequencing.Importantly,the off-target sites identified in vitro might differ from those in vivo ,which need to be further eval-uated by the applications of BLESS or other unbiased techniques such as those published during the revision of this work 43,44.Finally,we did not observe any overt signs of acute toxicity in mice at one to four weeks after virus administration.Although more studies are needed to further improve the SaCas9system for in vivo genome editing,such as assess-ing the long-term impact of Cas9and sgRNA expression,these findings suggest that in vivo genome editing using SaCas9has the potential to be highly efficient and specific.

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A L T (I U l –1)

A S T (I U

l –

)

Days post injection

T o t a l b i l i r u b i n (m g d l –1)

A l b u m i n (g d l –1)

AAV: Pcsk9-sg1

AAV: EGFP

Uninjected

Figure 5|Liver function tests and toxicity examination in injected animals.a ,Histological analysis of the liver at 1week post-injection by haematoxylin and eosin stain.Scale bars,10m m.b ,Liver function tests in Pcsk9-targeted (both Pcsk9-sg1and Pcsk9-sg2;231011total genome copies,n $4),

TBG–EGFP-injected (231011total genome copies,n 53),and uninjected (n 55)animals.Dashed lines show the upper and lower ranges of normal value in mice where applicable.

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Supplementary Information is available in the online version of the paper. Acknowledgements 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 T32GM007753from 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-CA133404from the National Institutes of Health,and PO1-CA42063from the National Cancer Institute,and partially by Cancer Center Support(core)grant P30-CA14051from 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:// https://www.wendangku.net/doc/ea1164142.html,.

Author Contributions F.A.R.and F.Z.conceived this study.F.A.R.,L.C.,W.X.Y.and F.Z. designed and performed the experiments with help from all authors.F.A.R.,J.S.G.,O.S., K.S.M.,E.V.K.and F.Z.contributed to analysis of Cas9orthologues,crRNA and tracrRNA, and PAM.A.J.K.,F.A.R.,X.W.,and P.A.S.led ChIP and computational analysis and validation.F.A.R.,W.X.Y.and L.C.performed BLESS and targeted sequencing of BLESS-identified off-target sites,and D.A.S.contributed computational analysis of BLESS data.W.X.Y.,F.A.R.,L.C.and B.Z.contributed animal data.W.X.Y.,F.A.R.,L.C., J.S.G.,and F.Z.wrote the manuscript with help from all authors.

Author Information All reagents described in this manuscript have been deposited with Addgene(plasmid IDs61591,61592and61593).Source data are available online and deep sequencing data are available at Sequence Read Archive under BioProject accession number PRJNA274149.Reprints and permissions information is available at https://www.wendangku.net/doc/ea1164142.html,/reprints.The authors declare competing financial interests:details are available in the online version of the paper.Readers are welcome to comment on the online version of the paper.Correspondence and requests for materials should be addressed to F.Z.(zhang@https://www.wendangku.net/doc/ea1164142.html,).

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6|N A T U R E|V O L000|00M O N T H2015

METHODS

No statistical methods were used to predetermine sample size.

In vitro transcription and cleavage assay.Cas9orthologues were human codon-optimized and synthesized by GenScript,and transfected into293FT cells as described below.Whole-cell lysates from293FT cells were prepared with lysis buffer(20mM HEPES,100mM KCl,5mM MgCl2,1mM DTT,5%glycerol,0.1%Triton X-100) supplemented with Protease Inhibitor Cocktail(Roche).T7-driven sgRNA was transcribed in vitro using custom oligonucleotides(Supplementary Information) and HiScribe T7In vitro Transcription Kit(NEB),following the manufacturer’s recommended protocol.The in vitro cleavage assay was carried out as follows:for a 20m l cleavage reaction,10m l of cell lysate was incubated with2m l cleavage buffer (100mM HEPES,500mM KCl,25mM MgCl2,5mM DTT,25%glycerol),1m g in vitro transcribed RNA and200ng EcoRI-linearized pUC19plasmid DNA or 200ng purified PCR amplicons from mammalian genomic DNA containing target sequence.After30min incubation,cleavage reactions were purified using QIAquick Spin Columns and treated with RNase A at final concentration of80ng m l21for 30min and analysed on a1%agarose E-Gel(Life Technologies).

In vitro PAM screen.Rho-independent transcriptional termination was predicted using the ARNold terminator search tool46,47.For the PAM library,a degenerate 7-bp sequence was cloned into a pUC19vector.For each orthologue,the in vitro cleavage assay was carried out as above with1m g T7-transcribed sgRNA and400ng pUC19with degenerate PAM.Cleaved plasmids were linearized by NheI,gel extracted,and ligated with Illumina sequencing adaptors.Barcoded and purified DNA libraries were quantified by Quant-iT PicoGreen dsDNA Assay Kit or Qubit 2.0Fluorometer(Life Technologies)and pooled in an equimolar ratio for sequenc-ing using the Illumina MiSeq Personal Sequencer(Life Technologies).MiSeq reads were filtered by requiring an average Phred quality(Q score)of at least23,as well as perfect sequence matches to barcodes.For reads corresponding to each orthologue, the degenerate region was extracted.All extracted regions were then grouped and analysed with Weblogo45.

Cell culture and transfection.Human embryonic kidney293FT(Life Technologies), Neuro-2a(N2a),and Hepa1-6(ATCC)cell lines were maintained in Dulbecco’s modified Eagle’s medium(DMEM)supplemented with10%FBS(HyClone),2mM GlutaMAX(Life Technologies),100U ml21penicillin,and100m g ml21strep-tomycin at37u C with5%CO2incubation.

Cells were seeded into24-well plates(Corning)one day before transfection at a density of240,000cells per well,and transfected at70–80%confluency using Lipofectamine2000(Life Technologies)following the manufacturer’s recom-mended protocol.For each well of a24-well plate,a total of500ng DNA was used. For ChIP and BLESS,a total of4.5million cells are seeded the day before transfec-tion into a100-mm plate,and a total of20m g DNA was used.

DNA isolation from cells and tissue.Genomic DNA was extracted using the QuickExtract DNA Extraction Solution(Epicentre).Briefly,pelleted cells were resuspended in QuickExtract solution and incubated at65u C for15min,68u C for15min,and98u C for10min(ref.8).Genomic liver DNA was extracted from bulk tissue fragments using a microtube bead mill homogenizer(Beadbug,Denville Scientific)by homogenizing approximately30–50mg of tissue in600m l of DPBS (Gibco).The homogenate was then centrifuged at2,000to3,000g for5min at4u C and the pellet was resuspended in300–600m l QuickExtract DNA Extraction Solu-tion(Epicentre)and incubated as above.

Indel analysis and guide:target base-pairing mismatch search.Indel analyses by SURVEYOR assay and targeted deep sequencing were carried out and analysed as previously described8,22.The methods for identification of potential off-target sites for SpCas9based on Watson–Crick base-pairing mismatch between guide RNA and target DNA has been previously described22,and adapted for SaCas9by con-sidering NNGRR for possible off-target PAMs.Alignment was manually adjusted to allow for insertion and deletion mismatches in the guide:target heteroduplex29,30. Chromatin immunoprecipitation and analysis.Cells were passaged at24h post-transfection into a150-mm dish,and fixed for ChIP processing at48h post-transfection.For each condition,10million cells are used for ChIP input,following experimental protocols and analyses as previously described31with the following modifications:instead of pairwise peak-calling,ChIP peaks were only required to be enriched over both‘empty’controls(dSpCas9only,dSaCas9only)as well as the other Cas9/other sgRNA sample(for example,SpCas9/EMX-sg2peaks must be enriched over SaCas9/EMX-sg1peaks in addition to the empty controls).This was done to avoid filtering out of real peaks present in two related samples as much as possible.

To identify off-targets ranked by motif or sequence similarity to guide,motif scores for ChIP peaks were calculated as follows:for a given ChIP peak,the100-nucleotide interval around the peak summit,the target sequence,and a given sgRNA guide region of length L,the query,an alignment score is calculated for every subsequence of L in the target.The subsequence with the highest score is reported as the best match to the query.For each subsequence alignment,the score calcula-tion begins at the59end of the query.For each position in the alignment,1is added or subtracted for match or mismatch between the query and target,respectively.If the score becomes negative,it is set to0and the calculation continued for the remainder of the alignment.The score at the39end of the query is reported as the final score for the alignment.MACS scores5210log(P value relative to the empty control)are determined as previously described48.For unbiased determination of PAM from ChIP peaks,the peaks were analysed for the best match by motif score to the guide region only within50nucleotides of the peak summit;the alignment was extended for10nucleotides at the39end and visualized using Weblogo45. To calculate the motif score threshold at which false discovery rate,0.1for each sample,100-nucleotide sequences centred around peak summits were shuf-fled while preserving dinucleotide frequency.The best match by motif score to the guide1PAM(NGG for SpCas9,NNGRRT for SaCas9)in these shuffled sequences was then found.The score threshold for false discovery rate,0.1was defined as the score such that less than10%of shuffled peaks had a motif score above that score threshold.

BLESS for DSB detection.Cells are harvested at24h post-transfection,then processed as previously described33with the following alterations:a total of10 million cells are fixed for nuclei isolation and permeabilization,and treated with Proteinase K for4min at37u C before inactivation with PMSF.All deproteinized nuclei are used for DSB labelling with100mM of annealed proximal linkers over-night.After Proteinase K digestion of labelled nuclei,chromatin was mechanic-ally sheared with a26G needle before sonication(BioRuptor,20min on high,50% duty cycle).20m g of sheared chromatin are captured on streptavidin beads,washed, and ligated to200mM of distal linker.Linker hairpins are then cleaved off with I-SceI digestion for1h at37u C,and products PCR-enriched for18cycles before proceeding to library preparation with TruSeq Nano LT Kit(Illumina).For the negative control,cells mock transfected with Lipofectamine2000and pUC19DNA were parallel processed through the assay.

BLESS analysis.Fastq files were demultiplexed,and30-bp genomic sequences were separated from the BLESS ligation handles for alignment.Bowtie was used to map the genomic sequences to hg19or mm9,allowing for a maximum of2mis-matches.Following alignment,reads from all bio-replicates for an individual sample were first pooled,and then nearest neighbour clustering was performed with a30-bp moving window to identify regions of enrichment across the genome.Within each cluster,the pairwise distance was calculated between all forward and reverse read strand mappings(Extended Data Fig.7b,c).Pairwise distance distributions were used to filter out wide and poorly defined DSB clusters from the well-defined DSB clusters characteristically found at Cas9-induced cleavage sites(see Supplementary Information).Finally,we adjusted the count of predicted Cas9-induced DSBs at a given locus by using a binomial model to calculate the maximum-likelihood esti-mate of peak enrichment in the Cas9-sgRNA treated sgRNAs given BLESS mea-surements from an untreated negative control.After the maximum-likelihood estimate calculation,a list of loci ranked by their DSB scores could be obtained and plotted(Fig.3b,Extended Data Fig.8).Additional descriptions can be found in Supplementary Information.

The top-ranking,30sites from the list of Cas9induced DSB clusters were sequenced for indel formation(Extended Data Fig.8;validated targets in Fig.3d). Within these loci,PAMs and regions of target homology were identified by first searching all PAM sites within a650bp window around the DSB cluster,then selecting the adjacent sequence with fewest mismatches to the target sequence. Code availability.BLESS analysis code is available at https://https://www.wendangku.net/doc/ea1164142.html,/fengz-hanglab/BLESS.

Virus production and titration.For in-house viral production,293FT cells(Life Technologies)were maintained as described above in150mm plates.For each transfection,8m g of pAAV8serotype packaging plasmid,10m g of pDF6helper plasmid,and6m g of AAV2plasmid carrying the construct of interest were added to1mL of serum-free DMEM.125m l of PEI‘‘Max’’solution(1mg ml–1,pH57.1) was then added to the mixture and incubated at room temperature for5to10s. After incubation,the mixture was added to20ml of warm maintenance media and applied to each dish to replace the old growth media.Cells were harvested between 48h and72h post transfection by scraping and pelleting by centrifugation.The AAV2/8(AAV2inverted terminal repeat(ITR)vectors pseudo-typed with AAV8 capsid)viral particles were then purified from the pellet according to a previously published protocol49.

High titre and purity viruses were also produced by vector core facilities at Children’s Hospital Boston and Massachusetts Eye and Ear Infirmary(MEEI).These AAV vectors were then titred by real-time qPCR using a customized TaqMan probe against the transgene,and all viral preparations were titre-matched across different batches and production facilities before experiments.The purity of AAV vector was further verified by SDS–PAGE.

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Animal injection and processing.All mice cohorts were maintained at animal facility with standard diet and housing following IRB-approved protocols.AAV vector was delivered to5–6week old male C57/BL6mice intravenously via lateral tail vein injection.All dosages of AAV were adjusted to100m l or200m l with sterile phosphate buffered saline(PBS),pH7.4(Gibco)before the injection.Animals were not immunosuppressed or otherwise handled differently before injection or during the course of the experiment except the pre-bleed fasting as noted below.The animals were randomized to the different experimental conditions,with the inves-tigator not blinded to the assignments.

To track the serum levels of Pcsk9and total cholesterol,animals were fasted overnight for12h before blood collection by saphenous vein bleeds(no more than 100m l or10%of total blood volume per week).Multiple bleeds were made before tail vein delivery of AAV vector or control to collect pre-injection samples and to habituate the animals to handling during the procedure.After the blood was allowed to clot at room temperature,the serum was separated by centrifugation and stored at–20u C for subsequent analysis.For terminal procedures to collect liver tissue and larger serum volumes for chemistry panels,mice were euthanized by carbon dioxide inhalation.Subsequently,blood was collected via cardiac puncture.Trans-cardial perfusion with30ml PBS removed the remaining blood,after which liver samples were collected.The median lobe of liver was removed and fixed in10% neutral buffered formalin for histological analysis,while the remaining lobes were sliced in small blocks of size less than13133mm3and frozen for subsequent DNA or protein extraction.Histology and serum analysis.Following tissue harvesting as described above, flash-frozen mouse liver samples were embedded in OCT compound(Tissue Tek, Cat#4583),snap-frozen,and stored at280u C before processing.Frozen tissues were cryosectioned at4m m in thickness and stained with Oil Red O following man-ufacturer’s recommended protocol.Liver histology was assessed by H&E staining sections of10%neutral buffer formalin fixed liver sections.

Serum levels of Pcsk9were determined by ELISA using the Mouse Proprotein Convertase9/PCSK9Quantikine ELISA Kit(MPC-900,R&D Systems),following the manufacturer’s instructions.Total cholesterol levels were measured using the Infinity Cholesterol Reagent(Thermo Fisher)per the manufacturer’s instructions. Serum ALT,AST,albumin and total bilirubin were measured by an Olympus AU5400(IDEXX Memphis,TN).

46.Gautheret,D.&Lambert,A.Direct RNA motif definition and identification from

multiple sequence alignments using secondary structure profiles.J.Mol.Biol.

313,1003–1011(2001).

47.Macke,T.J.et al.RNAMotif,an RNA secondary structure definition and search

algorithm.Nucleic Acids Res.29,4724–4735(2001).

48.Zhang,Y.et al.Model-based analysis of ChIP-seq(MACS).Genome Biol.9,R137

(2008).

49.Veldwijk,M.R.et al.Development and optimization of a real-time quantitative

PCR-based method for the titration of AAV-2vector stocks.Mol.Ther.6,272–278 (2002).

50.Zuker,M.Mfold web server for nucleic acid folding and hybridization prediction.

Nucleic Acids Res.31,3406–3415(2003).

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Extended Data Figure1|Selection of Type II CRISPR-Cas loci from eight bacterial species.a,Distribution of lengths for Cas9.600Cas9orthologues19. 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.

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Extended Data Figure 2|Cas9orthologue cleavage pattern in vitro .Stacked bar graph indicates the fraction of targets cleaved at 2,3,4,or 5bp upstream of PAM for each Cas9orthologue;most Cas9enzymes cleave stereotypically at 3bp upstream of PAM (red triangle).

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Extended Data Figure3|Test of Cas9orthologue activity in293FT cells. a,SURVEYOR assays showing indel formation at human endogenous loci from co-transfection of Cas9orthologues 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,SaCas9generates indels efficiently for a multiple targets.c,Box-whisker plot of indel formation as a function of SaCas9guide length L,with unaltered guides (perfect match of L nucleotides,grey bars)or replacement of the59-most base of guide with guanine(G1L21nucleotides,blue bars)(n58guides).

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Extended Data Figure4|Optimization of SaCas9sgRNA scaffold in mammalian cells.a,Schematic of the Staphylococcus aureus subspecies aureus CRISPR locus.b,Schematic of SaCas9sgRNA with21-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. SaCas9cleaves targets with varying repeat:anti-repeat lengths in c,HEK293FT and d,Hepa1-6cell lines.(n53,error bars show s.e.m.)

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Extended Data Figure5|Genome-wide binding by Cas9-chromatin immunoprecipitation(dCas9-ChIP).a,Unbiased identification of PAM motif for dSaCas9and dSpCas9.Peaks were analysed for the best match by motif score to the guide region only within50nucleotides of the peak summit.The alignment was extended for10nucleotides at the39end 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 dSaCas9and dSpCas9.Position1on x axis indicates the first base of PAM.

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Extended Data Figure6|Indel measurements at candidate off-target sites based on ChIP.Indels at top off-target sites predicted by dCas9-ChIP for each Cas9and 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 Cas9enzymes.

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Extended Data Figure7|Analysis pipeline of sequencing data from BLESS. 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 than6bp(dashed vertical line)are indicated over histogram plots.

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Extended Data Figure8|Indel measurements at off-target sites based on DSB scores.List of top off-target sites ranked by DSB scores for each Cas9and 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 Cas9enzymes. N.D.,not determined.

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Extended Data Figure9|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 SaCas9and SpCas9,respectively.Lines connect the common targets(EMX1)and off-targets between the two Cas9enzymes.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.

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Extended Data Figure10|SaCas9targeting Apob locus in the mouse liver. 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 AAV2/8carrying thyroxine-binding globulin(TBG)promoter-driven SaCas9and

U6-driven guide at231011total genome copies(n51animal each).d,Oil-red staining of liver tissue from AAV-or saline-injected animals.Male C56BL/6 mice were injected at8weeks of age and analysed4weeks post injection.

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