Double Nicking by RNA-Guided CRISPR Cas9for Enhanced
Genome Editing Specificity
F.Ann Ran,1,2,3,4,5,11Patrick D.Hsu,1,2,3,4,5,11Chie-Yu Lin,1,2,3,4,6Jonathan S.Gootenberg,1,2,3,4
Silvana Konermann,1,2,3,4Alexandro E.Trevino,1David A.Scott,1,2,3,4Azusa Inoue,7,8,9,10Shogo Matoba,7,8,9,10 Yi Zhang,7,8,9,10and Feng Zhang1,2,3,4,*
1Broad Institute of MIT and Harvard,7Cambridge Center,Cambridge,MA02142,USA
2McGovern Institute for Brain Rearch
3Department of Brain and Cognitive Sciences
4Department of Biological Engineering
Massachutts Institute of Technology,Cambridge,MA02139,USA
5Department of Molecular and Cellular Biology,Harvard University,Cambridge,MA02138,USA
重庆在职研究生
6Harvard/MIT Division of Health Sciences and Technology
7Howard Hughes Medical Institute
字母表 26个
8Program in Cellular and Molecular Medicine
9Department of Genetics
10Harvard Stem Cell Institute
Harvard Medical School,Boston,MA02115,USA
month什么意思11The authors contributed equally to this work
*Correspondence:zhang@broadinstitute
dx.doi/10.ll.2013.08.021
SUMMARY
Targeted genome editing technologies have enabled a broad range of rearch and medical applications. The Cas9nuclea from the microbial CRISPR-Cas system is targeted to specific genomic loci by a20 nt guide quence,which can tolerate certain mis-matches to the DNA target and thereby promote undesired off-target mutagenesis.Here,we describe an approach that combines a Cas9nicka mutant with paired guide RNAs to introduce targeted dou-ble-strand breaks.Becau individual nicks in the genome are repaired with highfidelity,simultaneous nicking via appropriately offt guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bas for target cleavage.We demonstrate that using paired nicking can reduce off-target activity by50-to 1,500-fold in cell lines and to facilitate gene knockout in mou zygotes without sacrificing on-target cleav-age efficiency.This versatile strategy enables a wide variety of genome editing applications that require high specificity.
INTRODUCTION
The ability to perturb the genome in a preci and targeted fashion is crucial for understanding genetic contributions to biology and dia.Genome engineering of cell lines or animal models has
traditionally been accomplished through random mutagenesis or low-efficiency gene targeting.To facilitate genome editing,programmable quence-specific DNA nuclea technologies have enabled targeted modification of endogenous genomic quences with high efficiency,particu-larly in species that have proven traditionally genetically intrac-table(Carlson et al.,2012;Geurts et al.,2009;Takasu et al., 2010;Watanabe et al.,2012).The RNA-guided Cas9nucleas from the microbial CRISPR(clustered regularly interspaced short palindromic repeat)-Cas systems are robust and versatile tools for stimulating targeted double-stranded DNA breaks(DSBs)in eukaryotic cells(Chang et al.,2013;Cho et al.,2013;Cong et al.,2013;Deltcheva et al.,2011;Deveau et al.,2010;Friedland et al.,2013;Gratz et al.,2013;Horvath and Barrangou,2010; Jinek et al.,2013;Mali et al.,2013b;Wang et al.,2013),where the resulting cellular repair mechanisms—nonhomologous end-joining(NHEJ)or homology-directed repair(HDR)pathways—can be exploited to induce error-prone or defined alterations (Hsu and Zhang,2012;Perez et al.,2008;Urnov et al.,2010). The Cas9nuclea from Streptococcus pyogenes can be directed by a chimeric single-guide RNA(sgRNA)(Jinek et al., 2012)to any genomic locus followed by a50-NGG protospacer-adjacent motif(PAM).A20nt guide quence within the sgRNA directs Cas9to the genomic target via Watson-Crick ba pairing and can be easily programmed to target a desired genomic locus (Deltcheva et al.,2011;Deveau et al.,2010;Gasiunas et al.,2012; Jinek et al.,2012).Recent studies of Cas9specificit
y have demon-strated that,although each ba within the20nt guide quence contributes to overall specificity,multiple mismatches between the guide RNA and its complementary target DNA quence can be tolerated depending on the quantity,position,and ba identity of mismatches(Cong et al.,2013;Fu et al.,2013;Hsu et al.,2013;Jiang et al.,2013),leading to potential off-target
DSBs and indel formation.The unwanted mutations can poten-tially limit the utility of Cas9for genome editing applications that require high levels of precision,such as generation of isogenic cell lines for testing causal genetic variations (Soldner et al.,2011)or in vivo and ex vivo genome-editing-bad therapies.To improve the specificity of Cas9-mediated genome editing,we developed a strategy that combines the D10A mutant nicka version of Cas9(Cas9n)(Cong et al.,2013;Gasiunas et al.,2012;Jinek et al.,2012)with a pair of offt sgRNAs complementary to opposite strands of the target site.Whereas nicking of both DNA strands by a pair of Cas9nickas leads to site-specific DSBs and NHEJ,individual nicks are predominantly repaired by the high-fidelity ba excision repair pathway (BER)(Dianov and Husad
¨bscher,2013).A paired nicka strategy was described while this manuscript was under review,which suggests the possibility for engineering a system to ameliorate off-target activity (Mali et al.,2013a ).In a manner analogous to dimeric zinc finger nucle-as (ZFNs)(Miller et al.,2007;Port
eus and Baltimore,2003;Sander et al.,2011;Wood et al.,2011)and transcription-acti-vator-like effector nucleas (TALENs)(Boch et al.,2009;Chris-tian et al.,2010;Miller et al.,2011;Moscou and Bogdanove,2009;Reyon et al.,2012;Sanjana et al.,2012;Wood et al.,2011;Zhang et al.,2011),wherein DNA cleavage requires syner-gistic interaction of two independent specificity-encoding DNA-binding modules directing FokI nuclea monomers,this double-nicking strategy minimizes off-target mutagenesis
by
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B Figure 1.Effect of Guide Sequence Exten-sion on Cas9Activity
(A)Cas9with matching or mismatching sgRNA quences targeting a locus (target 1)within the human EMX1gene.
(B)SURVEYOR assay gel showing comparable modification of target 1by sgRNAs bearing 20and 30nt long guide quences.
(C)Northern blot showing that extended sgRNAs are largely procesd to 20nt guide-length sgRNAs in HEK293FT cells.
each individual Cas9n-sgRNA complex while maintaining on-target modification rates similar to tho of wild-type Cas9.Here,we define crucial parameters for the lection of sgRNA pairs that facilitate effective double nicking,compare the specificity of wild-type Cas9and Cas9n with double nicking,and demonstrate a variety of experimental applications that can be achieved using double nicking in cells as well as in mou zygotes.RESULTS
Extension of Guide Sequence Does Not Improve Cas9Targeting Specificity
Cas9targeting is facilitated by ba pair-ing between the 20nt guide quence
within the sgRNA and the target DNA (Deltcheva et al.,2011;Deveau et al.,2010;Gasiunas et al.,2012;Jinek et al.,2012).We reasoned that cleavage specificity might be improved by increasing the length of ba pairing between the guide RNA and its target locus.To test this,we generated U6-d
riven expression casttes (Hsu et al.,2013)to express three sgRNAs with 20(sgRNA 1)or 30nt guide quences (sgRNAs 2and 3)targeting a locus within the human EMX1gene (Figure 1A).
We and others have previously shown that,although single-ba mismatches between the PAM-distal region of the guide quence and target DNA are well tolerated by Cas9,multiple mismatches in this region can significantly affect on-target activ-ity (Fu et al.,2013;Hsu et al.,2013;Mali et al.,2013a;Pattanayak et al.,2013).To determine whether additional PAM-distal bas (21–30)could influence overall targeting specificity,we designed sgRNAs 2and 3to contain additional bas consisting of either 10perfectly matched or 8mismatched bas (bas 21–28).Surprisingly,we obrved that the extended sgRNAs medi-ated similar levels of modification at the target locus in HEK293FT cells regardless of whether the additional bas were complementary to the genomic target (Figure 1B).Sub-quent northern blots revealed that the majority of both sgRNA 2and 3were procesd to the same length as sgRNA 1,which contains the same 20nt guide quence without additional ba-s (Figure 1C).
Cas9Nicka Generates Efficient NHEJ with Paired,Offt Guide RNAs
Given that extension of the guide quence failed to improve Cas9targeting specificity,we sought an alternative strategy for increasing the overall ba-pairing length between the guide quence and its DNA target.Cas9enzymes contain two conrved nuclea domains,HNH and RuvC,which cleave the DNA strand complementary and noncomplementary to the guide RNA,respectively.Mutations of the catalytic residues (D10A in RuvC and H840A in HNH)convert Cas9into DNA nickas (Cong et al.,2013;Gasiunas et al.,2012;Jinek et al.,2012).As single-strand nicks are preferentially repaired by the
high-fidelity BER pathway (Dianov and Hu
¨bscher,2013),we reasoned that two Cas9-nicking enzymes directed by a pair of sgRNAs targeting opposite strands of a target locus could mediate DSBs while minimizing off-target activity (Figure 2A).A number of factors may affect cooperative nicking leading to indel formation,including steric hindrance between two adjacent Cas9molecules or Cas9-sgRNA complexes,overhang type,
and
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Figure 2.Double Nicking Facilitates Effi-cient Genome Editing in Human Cells
(A)Schematic illustrating DNA double-stranded breaks using a pair of sgRNAs guiding Cas9D10A nickas (Cas9n).The D10A mutation renders Cas9able to cleave only the strand complemen-tary to the sgRNA;a pair of sgRNA-Cas9n com-plexes can nick both strands simultaneously.sgRNA offt is defined as the distance between the PAM-distal (50)ends of the guide quence of a given sgRNA pair;positive offt requires the sgRNA complementary to the top strand (sgRNA a)to be 50of the sgRNA complementary to the bottom strand (sgRNA b),which always creates a 50overhang.
(B)Efficiency of double-nicking-induced NHEJ as a function of the offt distance between two sgRNAs.Sequences for all sgRNAs ud can be found in Table S1.n =3;error bars show mean ±SEM.
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(C)Reprentative quences of the human EMX1locus targeted by Cas9n.sgRNA target sites and PAMs are indicated by blue and magenta bars,respectively.(Bottom)Selected quences showing reprentative indels.See also Tables S1and S2.
quence context;some of the may be characterized by testing multiple sgRNA pairs with distinct target quences and offts (the distance between the PAM-distal [50]ends of the guide quence of a given sgRNA pair).To systematically asss how sgRNA offts might affect subquent repair a
nd generation of in-dels,we first designed ts of sgRNA pairs targeted against the human EMX1genomic locus parated by a range of offt distances from approximately À200to 200bp to create both 50and 30
overhang products (Figure 2A and Table S1available online).We then assd the ability of each sgRNA pair with the D10A Cas9mutant (referred to as Cas9n;H840A Cas9mutant is referred to as Cas9H840A)to generate indels in human HEK 293FT cells.Robust NHEJ (up to 40%)was obrved for sgRNA pairs with offts from À4to 20bp,with modest indels forming in pairs offt by up to 100bp (Figure 2B,left).We subquently recapitulated the findings by testing similarly offt sgRNA pairs at two other genomic loci,DYRK1A and GRIN2B (Figure 2B,right).Of note,across all three loci examined,only sgRNA pairs creating 50overhangs with less than 8bp overlap between the guide quences (offt greater than À8bp)were able to mediate detectable indel formation (Figure 2C).
Importantly,each guide ud in the assays is able to effi-ciently induce indels when paired with wild-type Cas9(Table S1),indicating that the relative positions of the guide pairs are the most important parameters in predicting double-nicking activity.Becau Cas9n and Cas9H840A nick opposite strands of DNA,substitution of Cas9n with Cas9H840A with a given
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sgRNA pair should result in the inversion of the overhang type. For example,a pair of sgRNAs that will generate a50overhang with Cas9n should,in principle,generate the corresponding30 overhang instead.Therefore,sgRNA pairs that lead to the gener-ation of a30overhang with Cas9n might be ud with Cas9H840A to generate a50overhang.Further work will be needed to identify the necessary design rules for sgRNA pairing to allow double nicking by Cas9H840A.
Double Nicking Mediates Efficient Genome Editing with Improved Specificity
Having established that double nicking(DN)mediates high-effi-ciency NHEJ at levels comparable to tho induced by wild-type Cas9(Table S1),we next studied whether DN has improved specificity over wild-type Cas9by measuring their off-target ac-tivities.We co-delivered Cas9n with sgRNAs1and9,spaced by a+23bp offt,to target the human EMX1locus in HEK293FT cells(Figure3A).This DN configuration generated on-target indel levels similar to tho generated by
the wild-type Cas9paired with each sgRNA alone(Figure3B,left).Strikingly,unlike with wild-type Cas9,DN did not generate detectable modification at a previously validated sgRNA1off-target site,OT-4,by SUR-VEYOR assay(Hsu et al.,2013;Figure3B,right),suggesting that DN can potentially reduce the likelihood of off-target modifications.生日快乐英文怎么写
Using deep quencing to asss modification atfive different sgRNA1off-target loci(Figure3A),we obrved significant mutagenesis at all sites with wild-type Cas9+sgRNA1(Fig-ure3C).In contrast,cleavage by Cas9n at5off-target sites tested was barely detectable above background quencing error.Using the ratio of on-to off-target modification levels as a metric of specificity,we found that Cas9n with a pair of sgRNAs was able to achieve>100-fold greater specificity relative to wild-type Cas9with one of the sgRNAs(Figure3D).We conducted additional off-target analysis by deep quencing for two sgRNA pairs(offts of+16and+20bp)targeting the VEGFA locus,with similar results(Figure3E).DN at the off-target loci(Table S5) was able to achieve200-to>1,500-fold greater specificity than the wild-type Cas9(Figure3F and Table S1).Taken together,the results demonstrate that Cas9-mediated double nicking minimizes off-target mutagenesis and is suitable for genome editing with incread specificity.
Double Nicking Facilitates High-Efficiency Homology-Directed Repair,NHEJ-Mediated DNA Inrtion,
and Genomic Microdeletions
DSBs can stimulate homology-directed repair(HDR)to enable highly preci editing of genomic target sites.To evaluate DN-induced HDR,we targeted the human EMX1locus with pairs of sgRNAs offt byÀ3and+18bp(generating31and52bp 50overhangs),respectively,and introduced a single-stranded oligodeoxynucleotide(ssODN)bearing a Hin dIII restriction site as the HDR repair template(Figure4A).Each DN sgRNA pair successfully induced HDR at frequencies higher than tho of single-guide Cas9n nickas and comparable to tho of wild-type Cas9(Figure4B).Furthermore,genome editing in embry-onic stem cells or patient-derived induced pluripotent stem cells reprents a key opportunity for generating and studying new dia paradigms as well as developing new therapeutics. Becau single-nick approaches to inducing HDR in human embryonic stem cells(hESCs)have met with limited success (Hsu et al.,2013),we attempted DN in the HUES62hES cell line and obrved successful HDR(Figure4C).
To further characterize how offt sgRNA spacing affects the efficiency of HDR,we next tested in HEK293FT cells a t of sgRNA pairs in which the cleavage site of at least one sgRNA is situated near the site of recombination(overlapping with the HDR ssODN donor template arm).We obrved that sgRNA pairs generating50overhangs and having at least one nick occur-ring within22bp of the h
omology arm are able to induce HDR at levels comparable to tho of wild-type Cas9-mediated HDR and significantly greater than tho of single Cas9n-sgRNA nick-ing.In contrast,we did not obrve HDR with sgRNA pairs that generated30overhangs or double nicking of the same DNA strand(Figure4D).
The ability to create defined overhangs could enable preci inrtion of donor repair templates containing compatible over-hangs via NHEJ-mediated ligation(Maresca et al.,2013).To explore this alternative strategy for transgene inrtion,we tar-geted the EMX1locus with Cas9n and an sgRNA pair designed to generate a43bp50overhang near the stop codon and sup-plied a double-stranded oligonucleotide(dsODN)duplex with matching overhangs(Figure5A).The annealed dsODN inrt, containing multiple epitope tags and a restriction site,was suc-cessfully integrated into the target(1out of37screened by Sanger quencing of cloned amplicons).This ligation-bad strategy thus illustrates an effective approach for inrting dsODNs encoding short modifications such as protein tags or recombination sites into an endogenous locus.
Additionally,we targeted combinations of sgRNA pairs(four sgRNAs per combination)to the DYRK1A locus in HEK293FT cells to facilitate genomic microdeletions.We generated a t of sgRNAs to mediate0.5kb,1kb,2kb,and6kb deletions(Fig-ure5B and Table S2;sgRNAs32,33,and54–61)and verifi
ed successful multiplex nicking-mediated deletion over the ranges via PCR screen of predicted deletion sizes.
Figure3.Double Nicking Facilitates Efficient Genome Editing in Human Cells
(A)Schematic illustrating Cas9n double nicking(red arrows)the human EMX1locus.Five off-target loci with quence homology to EMX1target1were lected to screen for Cas9n specificity.
(B)On-target modification rate by Cas9n and a pair of sgRNAs is comparable to tho mediated by wild-type Cas9and single sgRNAs(left).Cas9-sgRNA1 complexes generate significant off-target mutagenesis,whereas no off-target locus modification is detected with Cas9n(right).
(C)Five off-target loci of sgRNA1are examined for indel modifications by deep quencing of transfected HEK293FT cells.n=3;error bars show mean±SEM.
(D)Specificity comparison of Cas9n with double nicking and wild-type Cas9with sgRNA alone at the off-target sites.Specificity ratio is calculated as on-target/ off-target modification rates.n=3;error bars show mean±SEM.
(E and F)Double nicking minimizes off-target modification at two additional human VEGFA loci while
maintaining high specificity(on/off-target modification ratio). n=3;error bars show mean±SEM.
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Figure4.Double Nicking Allows Inrtion into the Genome via HDR in Human Cells
(A)Schematic illustrating HDR mediated via a single-stranded oligodeoxynucleotide(ssODN)template at a DSB created by a pair of Cas9n enzymes.A12nt quence(red),including a Hin dIII restriction site,is inrted into the EMX1locus at the position marked by the gray dashed lines;distances of Cas9n-mediated nicks from the HDR inrtion site are indicated on top in italics.
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(B)Restriction digest assay gel showing successful inrtion of Hin dIII cleavage sites by double-nicking-mediated HDR in HEK293FT cells.Top bands are unmodified template;bottom bands are HindIII cleavage product.
sunt什么意思(C)Double nicking promotes HDR in the HUES62human embryonic stem cell line.HDR frequencies are determined by deep quencing.n=3;error bars show mean±SEM.
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