The CRISPR revolution in genome editing

Martin Jinek Department of Biochemistry, University of Zurich

Transformative capabilities for cellular engineering and analysis for biotechnology and biomedical research

Founders

Rachel Haurwitz President and CEO

Jennifer A. Doudna Professor

UC Berkeley

James M. Berger Professor

Johns Hopkins

Martin Jinek Assist. Professor

University of Zurich

The CRISPR revolution

• CRISPR-Cas9 genome editing is simple, fast, precise and cheap

• It is a powerful method for basic research, biotechnology and biomedicine

• Thanks to CRISPR-Cas9, somatic gene therapy can become a reality

• For the first time, humans have a method for modifying their own DNA

2012: programming DNA cleavage using RNA

A Programmable Dual-RNA–GuidedDNA Endonuclease in AdaptiveBacterial ImmunityMartin Jinek,1,2* Krzysztof Chylinski,3,4* Ines Fonfara,4 Michael Hauer,2†Jennifer A. Doudna,1,2,5,6‡ Emmanuelle Charpentier4‡

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systemsprovide bacteria and archaea with adaptive immunity against viruses and plasmids by usingCRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in asubset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA)forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introducedouble-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence,the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-likedomain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as asingle RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals afamily of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights thepotential to exploit the system for RNA-programmable genome editing.

Bacteria and archaea have evolved RNA-mediated adaptive defense systems calledclustered regularly interspaced short pal-

indromic repeats (CRISPR)/CRISPR-associated(Cas) that protect organisms from invading vi-ruses and plasmids (1–3). These defense systemsrely on small RNAs for sequence-specific de-tection and silencing of foreign nucleic acids.CRISPR/Cas systems are composed of cas genesorganized in operon(s) and CRISPR array(s) con-sisting of genome-targeting sequences (calledspacers) interspersed with identical repeats (1–3).CRISPR/Cas-mediated immunity occurs in threesteps. In the adaptive phase, bacteria and archaeaharboring one or more CRISPR loci respond toviral or plasmid challenge by integrating shortfragments of foreign sequence (protospacers)into the host chromosome at the proximal endof the CRISPR array (1–3). In the expression andinterference phases, transcription of the repeat-spacer element into precursor CRISPR RNA(pre-crRNA) molecules followed by enzymatic

cleavage yields the short crRNAs that can pairwith complementary protospacer sequences ofinvading viral or plasmid targets (4–11). Tar-get recognition by crRNAs directs the silencingof the foreign sequences by means of Cas pro-teins that function in complex with the crRNAs(10, 12–20).

There are three types of CRISPR/Cas systems(21–23). The type I and III systems share someoverarching features: specialized Cas endo-nucleases process the pre-crRNAs, and oncemature,each crRNA assembles into a large multi-Casprotein complex capable of recognizing andcleaving nucleic acids complementary to thecrRNA. In contrast, type II systems process pre-crRNAs by a different mechanism in which atrans-activating crRNA (tracrRNA) complemen-tary to the repeat sequences in pre-crRNA triggersprocessing by the double-stranded (ds) RNA-specific ribonuclease RNase III in the presenceof the Cas9 (formerly Csn1) protein (fig. S1)(4, 24). Cas9 is thought to be the sole proteinresponsible for crRNA-guided silencing of for-eign DNA (25–27).

We show here that in type II systems, Cas9proteins constitute a family of enzymes that re-quire a base-paired structure formed betweenthe activating tracrRNA and the targeting crRNAto cleave target dsDNA. Site-specific cleavage oc-curs at locations determined by both base-pairingcomplementarity between the crRNA and the tar-get protospacer DNA and a short motif [referredto as the protospacer adjacent motif (PAM)] jux-taposed to the complementary region in the tar-get DNA. Our study further demonstrates thatthe Cas9 endonuclease family can be programmedwith singleRNAmolecules to cleave specificDNAsites, thereby raising the exciting possibility of

developing a simple and versatile RNA-directedsystem to generate dsDNA breaks for genometargeting and editing.

Cas9 is a DNA endonuclease guided bytwo RNAs. Cas9, the hallmark protein of type IIsystems, has been hypothesized to be involvedin both crRNA maturation and crRNA-guidedDNA interference (fig. S1) (4, 25–27). Cas9 isinvolved in crRNA maturation (4), but its directparticipation in target DNA destruction has notbeen investigated. To test whether and how Cas9might be capable of target DNA cleavage, weused an overexpression system to purify Cas9protein derived from the pathogen Streptococcuspyogenes (fig. S2, see supplementary materialsand methods) and tested its ability to cleave a plas-mid DNA or an oligonucleotide duplex bearinga protospacer sequence complementary to a ma-ture crRNA, and a bona fide PAM.We found thatmature crRNA alone was incapable of directingCas9-catalyzed plasmid DNA cleavage (Fig. 1Aand fig. S3A). However, addition of tracrRNA,which can pair with the repeat sequence of crRNAand is essential to crRNA maturation in this sys-tem, triggered Cas9 to cleave plasmid DNA (Fig.1A and fig. S3A). The cleavage reaction requiredboth magnesium and the presence of a crRNAsequence complementary to the DNA; a crRNAcapable of tracrRNAbase pairing but containinga noncognate target DNA-binding sequence didnot support Cas9-catalyzed plasmid cleavage(Fig. 1A; fig. S3A, compare crRNA-sp2 tocrRNA-sp1; and fig. S4A). We obtained similarresults with a short linear dsDNA substrate (Fig.1B and fig. S3, B andC). Thus, the trans-activatingtracrRNA is a small noncoding RNAwith two crit-ical functions: triggering pre-crRNA processingby the enzyme RNase III (4) and subsequently ac-tivating crRNA-guided DNA cleavage by Cas9.

Cleavage of both plasmid and short lineardsDNA by tracrRNA:crRNA-guided Cas9 is site-specific (Fig. 1, C to E, and fig. S5, A and B).Plasmid DNA cleavage produced blunt ends ata position three base pairs upstream of the PAMsequence (Fig. 1, C and E, and fig. S5, A and C)(26). Similarly, within short dsDNA duplexes,the DNA strand that is complementary to thetarget-binding sequence in the crRNA (the com-plementary strand) is cleaved at a site three basepairs upstream of the PAM (Fig. 1, D and E, andfig. S5, B and C). The noncomplementary DNAstrand is cleaved at one or more sites within threeto eight base pairs upstream of the PAM. Furtherinvestigation revealed that the noncomplementarystrand is first cleaved endonucleolytically andsubsequently trimmed by a 3′-5′ exonuclease ac-tivity (fig. S4B). The cleavage rates by Cas9 un-der single-turnover conditions ranged from 0.3 to1 min−1, comparable to those of restriction endo-nucleases (fig. S6A), whereas incubation of wild-type (WT) Cas9-tracrRNA:crRNA complex witha fivefold molar excess of substrate DNA pro-vided evidence that the dual-RNA–guided Cas9is a multiple-turnover enzyme (fig. S6B). In

RESEARCHARTICLE

1Howard Hughes Medical Institute (HHMI), University of Cali-fornia, Berkeley, CA 94720, USA. 2Department of Molecularand Cell Biology, University of California, Berkeley, CA 94720,USA. 3Max F. Perutz Laboratories (MFPL), University of Vienna,A-1030 Vienna, Austria. 4The Laboratory for Molecular Infec-tion Medicine Sweden, Umeå Centre for Microbial Research,Department of Molecular Biology, Umeå University, S-90187Umeå, Sweden. 5Department of Chemistry, University of Cali-fornia, Berkeley, CA 94720, USA. 6Physical Biosciences Divi-sion, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA.

*These authors contributed equally to this work.†Present address: Friedrich Miescher Institute for BiomedicalResearch, 4058 Basel, Switzerland.‡To whom correspondence should be addressed. E-mail:doudna@berkeley.edu (J.A.D.); emmanuelle.charpentier@mims.umu.se (E.C.)

17 AUGUST 2012 VOL 337 SCIENCE www.sciencemag.org816

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DOI: 10.1126/science.1225829, 816 (2012);337 Science et al.Martin Jinek

Bacterial ImmunityGuided DNA Endonuclease in Adaptive−A Programmable Dual-RNA

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A Programmable Dual-RNA–GuidedDNA Endonuclease in AdaptiveBacterial ImmunityMartin Jinek,1,2* Krzysztof Chylinski,3,4* Ines Fonfara,4 Michael Hauer,2†Jennifer A. Doudna,1,2,5,6‡ Emmanuelle Charpentier4‡

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systemsprovide bacteria and archaea with adaptive immunity against viruses and plasmids by usingCRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in asubset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA)forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introducedouble-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence,the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-likedomain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as asingle RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals afamily of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights thepotential to exploit the system for RNA-programmable genome editing.

Bacteria and archaea have evolved RNA-mediated adaptive defense systems calledclustered regularly interspaced short pal-

indromic repeats (CRISPR)/CRISPR-associated(Cas) that protect organisms from invading vi-ruses and plasmids (1–3). These defense systemsrely on small RNAs for sequence-specific de-tection and silencing of foreign nucleic acids.CRISPR/Cas systems are composed of cas genesorganized in operon(s) and CRISPR array(s) con-sisting of genome-targeting sequences (calledspacers) interspersed with identical repeats (1–3).CRISPR/Cas-mediated immunity occurs in threesteps. In the adaptive phase, bacteria and archaeaharboring one or more CRISPR loci respond toviral or plasmid challenge by integrating shortfragments of foreign sequence (protospacers)into the host chromosome at the proximal endof the CRISPR array (1–3). In the expression andinterference phases, transcription of the repeat-spacer element into precursor CRISPR RNA(pre-crRNA) molecules followed by enzymatic

cleavage yields the short crRNAs that can pairwith complementary protospacer sequences ofinvading viral or plasmid targets (4–11). Tar-get recognition by crRNAs directs the silencingof the foreign sequences by means of Cas pro-teins that function in complex with the crRNAs(10, 12–20).

There are three types of CRISPR/Cas systems(21–23). The type I and III systems share someoverarching features: specialized Cas endo-nucleases process the pre-crRNAs, and oncemature,each crRNA assembles into a large multi-Casprotein complex capable of recognizing andcleaving nucleic acids complementary to thecrRNA. In contrast, type II systems process pre-crRNAs by a different mechanism in which atrans-activating crRNA (tracrRNA) complemen-tary to the repeat sequences in pre-crRNA triggersprocessing by the double-stranded (ds) RNA-specific ribonuclease RNase III in the presenceof the Cas9 (formerly Csn1) protein (fig. S1)(4, 24). Cas9 is thought to be the sole proteinresponsible for crRNA-guided silencing of for-eign DNA (25–27).

We show here that in type II systems, Cas9proteins constitute a family of enzymes that re-quire a base-paired structure formed betweenthe activating tracrRNA and the targeting crRNAto cleave target dsDNA. Site-specific cleavage oc-curs at locations determined by both base-pairingcomplementarity between the crRNA and the tar-get protospacer DNA and a short motif [referredto as the protospacer adjacent motif (PAM)] jux-taposed to the complementary region in the tar-get DNA. Our study further demonstrates thatthe Cas9 endonuclease family can be programmedwith singleRNAmolecules to cleave specificDNAsites, thereby raising the exciting possibility of

developing a simple and versatile RNA-directedsystem to generate dsDNA breaks for genometargeting and editing.

Cas9 is a DNA endonuclease guided bytwo RNAs. Cas9, the hallmark protein of type IIsystems, has been hypothesized to be involvedin both crRNA maturation and crRNA-guidedDNA interference (fig. S1) (4, 25–27). Cas9 isinvolved in crRNA maturation (4), but its directparticipation in target DNA destruction has notbeen investigated. To test whether and how Cas9might be capable of target DNA cleavage, weused an overexpression system to purify Cas9protein derived from the pathogen Streptococcuspyogenes (fig. S2, see supplementary materialsand methods) and tested its ability to cleave a plas-mid DNA or an oligonucleotide duplex bearinga protospacer sequence complementary to a ma-ture crRNA, and a bona fide PAM.We found thatmature crRNA alone was incapable of directingCas9-catalyzed plasmid DNA cleavage (Fig. 1Aand fig. S3A). However, addition of tracrRNA,which can pair with the repeat sequence of crRNAand is essential to crRNA maturation in this sys-tem, triggered Cas9 to cleave plasmid DNA (Fig.1A and fig. S3A). The cleavage reaction requiredboth magnesium and the presence of a crRNAsequence complementary to the DNA; a crRNAcapable of tracrRNAbase pairing but containinga noncognate target DNA-binding sequence didnot support Cas9-catalyzed plasmid cleavage(Fig. 1A; fig. S3A, compare crRNA-sp2 tocrRNA-sp1; and fig. S4A). We obtained similarresults with a short linear dsDNA substrate (Fig.1B and fig. S3, B andC). Thus, the trans-activatingtracrRNA is a small noncoding RNAwith two crit-ical functions: triggering pre-crRNA processingby the enzyme RNase III (4) and subsequently ac-tivating crRNA-guided DNA cleavage by Cas9.

Cleavage of both plasmid and short lineardsDNA by tracrRNA:crRNA-guided Cas9 is site-specific (Fig. 1, C to E, and fig. S5, A and B).Plasmid DNA cleavage produced blunt ends ata position three base pairs upstream of the PAMsequence (Fig. 1, C and E, and fig. S5, A and C)(26). Similarly, within short dsDNA duplexes,the DNA strand that is complementary to thetarget-binding sequence in the crRNA (the com-plementary strand) is cleaved at a site three basepairs upstream of the PAM (Fig. 1, D and E, andfig. S5, B and C). The noncomplementary DNAstrand is cleaved at one or more sites within threeto eight base pairs upstream of the PAM. Furtherinvestigation revealed that the noncomplementarystrand is first cleaved endonucleolytically andsubsequently trimmed by a 3′-5′ exonuclease ac-tivity (fig. S4B). The cleavage rates by Cas9 un-der single-turnover conditions ranged from 0.3 to1 min−1, comparable to those of restriction endo-nucleases (fig. S6A), whereas incubation of wild-type (WT) Cas9-tracrRNA:crRNA complex witha fivefold molar excess of substrate DNA pro-vided evidence that the dual-RNA–guided Cas9is a multiple-turnover enzyme (fig. S6B). In

RESEARCHARTICLE

1Howard Hughes Medical Institute (HHMI), University of Cali-fornia, Berkeley, CA 94720, USA. 2Department of Molecularand Cell Biology, University of California, Berkeley, CA 94720,USA. 3Max F. Perutz Laboratories (MFPL), University of Vienna,A-1030 Vienna, Austria. 4The Laboratory for Molecular Infec-tion Medicine Sweden, Umeå Centre for Microbial Research,Department of Molecular Biology, Umeå University, S-90187Umeå, Sweden. 5Department of Chemistry, University of Cali-fornia, Berkeley, CA 94720, USA. 6Physical Biosciences Divi-sion, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA.

*These authors contributed equally to this work.†Present address: Friedrich Miescher Institute for BiomedicalResearch, 4058 Basel, Switzerland.‡To whom correspondence should be addressed. E-mail:doudna@berkeley.edu (J.A.D.); emmanuelle.charpentier@mims.umu.se (E.C.)

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DNA cleavage can be programmed using engineered RNAs

Programmable one-protein, one-RNA system Targeting to a new site only requires designing a new guide RNA

loop

Jinek*, Chylinski* et al., Science (2012)

Jinek et al., eLife (2013)

RNA-guided genome editing using CRISPR-Cas9

DNA encoding Cas9 protein and sgRNA

CRISPR-Cas9 genome editing in 2013

Human cells Mali (Science); Cong (Science); Jinek (eLife), Cho (Nat Biotech); Hou (PNAS 2013), Esvelt (Nat Methods), Ran (Cell), Mali (Nat Biotech)

Mice/rats Cong (Science); Wang (Cell); Shen (Cell Res); Li W (Nat Biotech); Li D (Nat Biotech); Menke (Genetics), Yang (Cell), Hu (Cell Res), Fujii (NAR)

Zebrafish Hwang (Nat Biotech); Chang (Cell Res); Xiao (NAR); Jao (PNAS); Auer (Genome Res); Hruscha (Development)

Xenopus Nakayama (Genesis); Blitz (Genesis)

Drosophila Bassett (Cell Reports); Gratz (Genetics); Yu (Genetics), Sebo (Fly), Kondo (Genetics), Ren (PNAS)

Silkworm Wang (Cell Res)

Yeast di Carlo (NAR)

C. elegans Friedland (Nat Methods); Chiu (Genetics); Lo (Genetics), Chen (NAR), Dickinson (Nat Methods); Katic (Genetics)

Plants Li (Nat Biotech); Shan (Nat Biotech); Nekrasov (Nat Biotech); Mao (Mol Plant); Feng (Cell Res); Xie (Mol Plant), Jiang (NAR), Miao (Cell Res)

Bacteria Jiang (Nat Biotech); Bikard (NAR)

Mingming & Ningning

Niu et al., Cell (2014)

CRISPR-Cas9 genome editing in 2014

• two immunity genes targeted simultaneously (Rag1 and PPAR-gamma)• Cas9 mRNA and sgRNA injected into one-cell-stage embryos

CRISPR-Cas9 genome editing in 2015

tools has also been a limiting factor for the anal-ysis of gene functions in model organisms ofdevelopmental and regenerative biology. Effi-cient genome engineering to allow targetedgenome modifications in the germ lines of ani-mal models such as fruit flies (113, 114), zebra-fish (94, 115), nematodes (116), salamanders (117),and frogs (118, 119) is now possible with the de-velopment of the CRISPR-Cas9 technology. Thetechnology can also facilitate the generation ofmouse (120–122) and rat (123, 124) models bettersuited to pharmacological studies and the un-derstanding of human diseases, as well as pigs(125) andmonkeys (126). Overall, CRISPR-Cas9 isalready having a major impact on functionalgenomic experiments that can be conducted inthese model systems, which will advance thefield of experimental biology in ways not imag-ined even a few years ago.

Further development of the technology

A key property of Cas9 is its ability to bind toDNA at sites defined by the guide RNA sequenceand the PAM, allowing applications beyondpermanent modification of DNA. In particular,a catalytically deactivated version of Cas9 (dCas9)has been repurposed for targeted gene regula-tion on a genome-wide scale. Referred to asCRISPR interference (CRISPRi), this strategywas shown to block transcriptional elongation,RNA polymerase binding, or transcription factorbinding, depending on the site(s) recognized bythe dCas9–guide RNA complex. Demonstratedfirst in E. coli, whole-genome sequencing showedthat there were no detectable off-target effects(127). CRISPRi has been used to repress multipletarget genes simultaneously, and its effects arereversible (127–130).By generating chimeric versions of dCas9 that

are fused to regulatory domains, it has been pos-sible to use CRISPRi for efficient gene regulationin mammalian cells. Specifically, fusion of dCas9to effector domains including VP64 or KRABallowed stable and efficient transcriptional acti-vation or repression, respectively, in human andyeast cells (129). As observed in bacteria, site(s) ofregulation were defined solely by the coexpressedguideRNA(s) for dCas9. RNA-seq analysis showedthat CRISPRi-directed transcriptional repressionis highly specific. More broadly, these resultsdemonstrated that dCas9 can be used as amodular and flexible DNA-binding platform for

the recruitment of proteins to a target DNAsequence in a genome, laying the foundationfor future experiments involving genome-widescreening similar to those performed usingRNAi. The lack of CRISPR-Cas systems in eu-karyotes is an important advantage of CRISPRiover RNAi for various applications in whichcompetition with the endogenous pathways isproblematic. For example, using RNAi to si-lence genes that are part of the RNAi pathwayitself (i.e., Dicer, Argonaute) can lead to resultsthat are difficult to interpret due to multipledirect and indirect effects. In addition, any RNAsused to silence specific genes may compete withendogenous RNA-mediated gene regulation incells. With its ability to permanently change thegenetic code and to up- or down-regulate geneexpression at the transcriptional or posttranscrip-tional level, CRISPR-Cas9 offers a large versatilityin harnessing alternatives, whereas RNAi is mostlyrestricted to knocking down gene expression.Although RNAi has been improving over theyears, incomplete knockdowns or unpredictableoff-targeting are still reported bottlenecks of thistechnology, and future comparative analysesshould address the superiority of CRISPRi overRNAi in these aspects.The programmable binding capability of dCas9

can also be used for imaging of specific loci in livecells. An enhanced green fluorescent protein–tagged dCas9 protein and a structurally optimizedsgRNA were shown to produce robust imaging ofrepetitive and nonrepetitive elements in telomeresand coding genes in living cells (131). This CRISPRimaging tool has the potential to improve the cur-rent technologies for studying conformationaldynamics of native chromosomes in living cells,particularly ifmulticolor imaging can be developedusing multiple distinct Cas9 proteins. It may alsobe possible to couple fluorescent proteins or smallmolecules to the guide RNA, providing an orthog-onal strategy for multicolor imaging using Cas9.Novel technologies aiming to disrupt provi-

ruses may be an attractive approach to eliminat-ing viral genomes from infected individuals andthus curing viral infections. An appeal of thisstrategy is that it takes advantage of the primarynative functions of CRISPR-Cas systems as anti-viral adaptive immune systems in bacteria. Thetargeted CRISPR-Cas9 technique was shown toefficiently cleave and mutate the long terminalrepeat sites of HIV-1 and also to remove internal

viral genes from the chromosome of infected cells(132, 133).CRISPR-Cas9 is also a promising technology in

the field of engineering and synthetic biology. Amultiplex CRISPR approach referred to as CRISPRmwas developed to facilitate directed evolution ofbiomolecules (134). CRISPRm consists of the op-timization of CRISPR-Cas9 to generate quantita-tive gene assembly and DNA library insertioninto the fungal genomes, providing a strategy toimprove the activity of biomolecules. In addition,it has been possible to induce Cas9 to bind single-strandedRNA in a programmable fashion by usingshort DNA oligonucleotides containing PAM se-quences (PAMmers) to activate the enzyme, sug-gesting new ways to target transcripts withoutprior affinity tagging (135).A series of studies have reported the efficiency

with which the RNA-programmable S. pyogenesCas9 targets and cleaves DNA and have alsoaddressed the level of its specificity by moni-toring the ratio of off-site targeting (136–140).Off-site targeting is defined by the tolerance ofCas9 to mismatches in the RNA guide sequenceand is dependent on the number, position, anddistribution of mismatches throughout the en-tire guide sequence (136–140) beyond the initialseed sequence originally defined as the first 8to 12 nucleotides of the guide sequence prox-imal to the PAM (64) (Fig. 2). The amount ofCas9 enzyme expressed in the cell is an im-portant factor in tolerance to mismatches (138).High concentrations of the enzyme were re-ported to increase off-site targeting, whereaslowering the concentration of Cas9 increasesspecificity while diminishing on-target cleavageactivity (137). Several groups have developedalgorithmic tools that predict the sequence ofan optimal sgRNA with minimized off-target effects(for example, http://tools.genome-engineering.org,http://zifit.partners.org, and www.e-crisp.org)(141–145). The development of alternative genome-wide approaches that would also consider otherfeatures of the reaction, such as the thermody-namic properties of the sgRNA, may also in-crease the specificity of the design.Several studies of the CRISPR-Cas9 technol-

ogy relate to the specificity of DNA targeting(Fig. 4): a double-nicking approach consistingof using the nickase variant of Cas9 with a pairof offset sgRNAs properly positioned on thetarget DNA (146–148); an sgRNA-guided dCas9

fused to the FokI nuclease wheretwo fused dCas9-FokI monomerscan simultaneously bind target sitesat a defined distance apart (149, 150);and shorter sgRNAs truncated bytwo or three nucleotides at thedistal end relative to the PAM thatcan be used with the double nick-ing strategy to further reduce off-target activity (151). The first twomethods rely on Cas9 dimerizationsimilar to the engineered dimericZFNs and TALENs, with the princi-ple that two adjacent off-target bind-ing events and subsequent cleavage

1258096-6 28 NOVEMBER 2014 • VOL 346 ISSUE 6213 sciencemag.org SCIENCE

Cell linesHEK293U2OSK562

Model organismsMiceRatsFruit fliesNematodesArabidopsisSalamandersFrogsMonkeys

Biology Crop plantsRiceWheatSorghumTobacco

FungiKluyveromycesChlamydomonas

Biotechnology OrganoidshESCsiPSCs

Biomedicine

Fig. 5. Examples of cell types and organisms that have been engineered using Cas9.

RESEARCH | REVIEW

Doudna and Charpentier, Science (2014) Regalado, MIT Technology Review (2015)

are less likely to occur than a single off-targetcleavage (146–150). The latter method follows thereasoning according to which the 5′-end nucleo-tides of the sgRNAs are not necessary for theirfull activity; however, they may compensate formismatches at other positions along the guideRNA–target DNA interface, and thus shortersgRNAs may be more specific (151). Future ef-forts will focus on further developing the pre-cision of the technology, as well as increasingthe frequency of homology-directed repair rel-ative to nonhomologous end joining in orderto favor site-specific insertion of new geneticinformation.

Conclusions and perspectives

Our understanding of how genomes direct de-velopment, normal physiology, and disease inhigher organisms has been hindered by a lack ofsuitable tools for precise and efficient gene en-gineering. The simple two-component CRISPR-Cas9 system, usingWatson-Crick base pairing bya guide RNA to identify target DNA sequences, isa versatile technology that has already stimulatedinnovative applications in biology. Understandingthe CRISPR-Cas9 system at the biochemicaland structural level allows the engineering oftailored Cas9 variants with smaller size andincreased specificity. A crystal structure of thesmaller Cas9 protein from Actinomyces, for exam-ple, showed how natural variation created astreamlined enzyme, setting the stage for futureengineered Cas9 variants (77). A deeper analysisof the large panel of naturally evolving bacterialCas9 enzymes may also reveal orthologs withdistinct DNA binding specificity, will broadenthe choice of PAMs, and will certainly revealshorter variants more amenable for delivery inhuman cells.Furthermore, specific methods for delivering

Cas9 and its guide RNA to cells and tissuesshould benefit the field of human gene therapy.For example, recent experiments confirmed thatthe Cas9 protein-RNA complex can be intro-duced directly into cells using nucleofection orcell-penetrating peptides to enable rapid andtimed editing (89, 152), and transgenic organisms

that express Cas9 from inducible promoters arebeing tested. An exciting harbinger of futureresearch in this area is the recent demonstrationthat Cas9–guide RNA complexes, when injectedinto adult mice, provided sufficient editing in theliver to alleviate a genetic disorder (153). Under-standing the rates of homology-directed repairafter Cas9-mediated DNA cutting will advance thefield by enabling efficient insertion of new orcorrected sequences into cells and organisms. Inaddition, the rapid advance of the field has raisedexcitement about commercial applications ofCRISPR-Cas9.The era of straightforward genome editing

raises ethical questions that will need to beaddressed by scientists and society at large. Howcan we use this powerful tool in such a way as toensure maximum benefit while minimizing risks?It will be imperative that nonscientists understandthe basics of this technology sufficiently well tofacilitate rational public discourse. Regulatoryagencies will also need to consider how best tofoster responsible use of CRISPR-Cas9 technolo-gy without inhibiting appropriate research anddevelopment.The identification of the CRISPR-Cas9 tech-

nology underscores the way in which manyinventions that have advancedmolecular biologyand medicine emanated, through basic researchon natural mechanisms of DNA replication, re-pair, and defense against viruses. In many cases,key methodologies emerged from the study ofbacteria. The CRISPR-Cas9 technology originatedthrough a similar process: Once the mechanismunderlying how the CRISPR-Cas9 system workswas understood, it could be harnessed for applica-tions in molecular biology and genetics that werenot previously envisioned.

REFERENCES AND NOTES

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2. Y. S. Rong, K. G. Golic, Gene targeting by homologousrecombination in Drosophila. Science 288, 2013–2018 (2000).doi: 10.1126/science.288.5473.2013; pmid: 10856208

3. O. Smithies, R. G. Gregg, S. S. Boggs, M. A. Koralewski,R. S. Kucherlapati, Insertion of DNA sequences into the

human chromosomal beta-globin locus by homologousrecombination. Nature 317, 230–234 (1985). doi: 10.1038/317230a0; pmid: 2995814

4. K. R. Thomas, K. R. Folger, M. R. Capecchi, High frequencytargeting of genes to specific sites in the mammaliangenome. Cell 44, 419–428 (1986). doi: 10.1016/0092-8674(86)90463-0; pmid: 3002636

5. S. L. Mansour, K. R. Thomas, M. R. Capecchi, Disruption ofthe proto-oncogene int-2 in mouse embryo-derived stemcells: A general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 (1988). doi: 10.1038/336348a0; pmid: 3194019

6. N. Rudin, E. Sugarman, J. E. Haber, Genetic and physicalanalysis of double-strand break repair and recombinationin Saccharomyces cerevisiae. Genetics 122, 519–534 (1989).pmid: 2668114

7. A. Plessis, A. Perrin, J. E. Haber, B. Dujon, Site-specificrecombination determined by I-SceI, a mitochondrial group Iintron-encoded endonuclease expressed in the yeast nucleus.Genetics 130, 451–460 (1992). pmid: 1551570

8. P. Rouet, F. Smih, M. Jasin, Introduction of double-strandbreaks into the genome of mouse cells by expression of arare-cutting endonuclease. Mol. Cell. Biol. 14, 8096–8106(1994). pmid: 7969147

9. A. Choulika, A. Perrin, B. Dujon, J. F. Nicolas, Induction ofhomologous recombination in mammalian chromosomes byusing the I-SceI system of Saccharomyces cerevisiae. Mol.Cell. Biol. 15, 1968–1973 (1995). pmid: 7891691

10. G. Felsenfeld, D. R. Davies, A. Rich, Formation of a three-stranded polynucleotide molecule. J. Am. Chem. Soc. 79,2023–2024 (1957). doi: 10.1021/ja01565a074

11. A. Varshavsky, Discovering the RNA double helix andhybridization. Cell 127, 1295–1297 (2006). doi: 10.1016/j.cell.2006.12.008; pmid: 17190591

12. S. A. Strobel, L. A. Doucette-Stamm, L. Riba, D. E. Housman,P. B. Dervan, Site-specific cleavage of human chromosome 4mediated by triple-helix formation. Science 254, 1639–1642(1991). doi: 10.1126/science.1836279; pmid: 1836279

13. S. A. Strobel, P. B. Dervan, Single-site enzymatic cleavage ofyeast genomic DNA mediated by triple helix formation. Nature350, 172–174 (1991). doi: 10.1038/350172a0; pmid: 1848684

14. S. A. Strobel, P. B. Dervan, Site-specific cleavage of a yeastchromosome by oligonucleotide-directed triple-helixformation. Science 249, 73–75 (1990). doi: 10.1126/science.2195655; pmid: 2195655

15. A. F. Faruqi, M. M. Seidman, D. J. Segal, D. Carroll,P. M. Glazer, Recombination induced by triple-helix-targetedDNA damage in mammalian cells. Mol. Cell. Biol. 16,6820–6828 (1996). pmid: 8943337

16. G. Wang, D. D. Levy, M. M. Seidman, P. M. Glazer, Targetedmutagenesis in mammalian cells mediated by intracellulartriple helix formation. Mol. Cell. Biol. 15, 1759–1768 (1995).pmid: 7862165

17. Z. Sandor, A. Bredberg, Deficient DNA repair of triple helix-directed double psoralen damage in human cells. FEBS Lett.374, 287–291 (1995). doi: 10.1016/0014-5793(95)01133-Y;pmid: 7589555

18. J. Cho, M. E. Parks, P. B. Dervan, Cyclic polyamides forrecognition in the minor groove of DNA. Proc. Natl. Acad. Sci.U.S.A. 92, 10389–10392 (1995). doi: 10.1073/pnas.92.22.10389; pmid: 7479790

19. A. F. Faruqi, M. Egholm, P. M. Glazer, Peptide nucleic acid-targeted mutagenesis of a chromosomal gene in mouse cells.Proc. Natl. Acad. Sci. U.S.A. 95, 1398–1403 (1998).doi: 10.1073/pnas.95.4.1398; pmid: 9465026

20. J. M. Gottesfeld, L. Neely, J. W. Trauger, E. E. Baird, P. B. Dervan,Regulation of gene expression by small molecules. Nature 387,202–205 (1997). doi: 10.1038/387202a0; pmid: 9144294

21. J. Yang, S. Zimmerly, P. S. Perlman, A. M. Lambowitz,Efficient integration of an intron RNA into double-strandedDNA by reverse splicing. Nature 381, 332–335 (1996).doi: 10.1038/381332a0; pmid: 8692273

22. S. Zimmerly et al., A group II intron RNA is a catalyticcomponent of a DNA endonuclease involved in intronmobility. Cell 83, 529–538 (1995). doi: 10.1016/0092-8674(95)90092-6; pmid: 7585955

23. B. A. Sullenger, T. R. Cech, Ribozyme-mediated repair ofdefective mRNA by targeted, trans-splicing. Nature 371,619–622 (1994). doi: 10.1038/371619a0; pmid: 7935797

24. A. Jacquier, B. Dujon, An intron-encoded protein is active in agene conversion process that spreads an intron into amitochondrial gene. Cell 41, 383–394 (1985). doi: 10.1016/S0092-8674(85)80011-8; pmid: 3886163

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Human gene therapy

Screens for drug target ID Agriculture: crops, animals

Ecological vector control:mosquito sterilization, etc.

Viral gene disruption;pathogen gene disruption

Programmable RNA targeting

Synthetic biology;pathway engineering

The future of CRISPR-Cas9-mediated genome engineering

Fig. 6. Future applications in biomedicine and biotechnology. Potential developments includeestablishment of screens for target identification, human gene therapy by gene repair and genedisruption, gene disruption of viral sequences, and programmable RNA targeting.

RESEARCH | REVIEW

CRISPR-Cas9: Genetic engineering for the masses

• Easy sgRNA design • Fast • Cheap • Specific • Versatile • Scalable for genome-wide

screens • Multiplexable • Adaptable for transcriptional

and epigenetic control

Applications of Cas9-based genome editing

developed in recent years, enabling targeted and efficient modi-fication of a variety of eukaryotic and particularly mammalianspecies. Of the current generation of genome editing technolo-gies, the most rapidly developing is the class of RNA-guidedendonucleases known as Cas9 from the microbial adaptive im-mune system CRISPR (clustered regularly interspaced shortpalindromic repeats), which can be easily targeted to virtuallyany genomic location of choice by a short RNA guide. Here,we review the development and applications of the CRISPR-associated endonuclease Cas9 as a platform technology forachieving targeted perturbation of endogenous genomic ele-ments and also discuss challenges and future avenues for inno-vation.

Programmable Nucleases as Tools for Efficient andPrecise Genome EditingA series of studies by Haber and Jasin (Rudin et al., 1989; Plessiset al., 1992; Rouet et al., 1994; Choulika et al., 1995; Bibikovaet al., 2001; Bibikova et al., 2003) led to the realization that tar-

geted DNA double-strand breaks (DSBs) could greatly stimulategenome editing through HR-mediated recombination events.Subsequently, Carroll and Chandrasegaran demonstrated thepotential of designer nucleases based on zinc finger proteinsfor efficient, locus-specific HR (Bibikova et al., 2001, 2003).Moreover, it was shown in the absence of an exogenous homol-ogy repair template that localized DSBs can induce insertions ordeletion mutations (indels) via the error-prone nonhomologousend-joining (NHEJ) repair pathway (Figure 2A) (Bibikova et al.,2002). These early genome editing studies established DSB-induced HR and NHEJ as powerful pathways for the versatileand precise modification of eukaryotic genomes.To achieve effective genome editing via introduction of site-

specific DNA DSBs, four major classes of customizable DNA-binding proteins have been engineered so far: meganucleasesderived from microbial mobile genetic elements (Smith et al.,2006), zinc finger (ZF) nucleases based on eukaryotic transcrip-tion factors (Urnov et al., 2005; Miller et al., 2007), transcriptionactivator-like effectors (TALEs) from Xanthomonas bacteria(Christian et al., 2010; Miller et al., 2011; Boch et al., 2009; Mos-cou and Bogdanove, 2009), and most recently the RNA-guidedDNA endonuclease Cas9 from the type II bacterial adaptive im-mune system CRISPR (Cong et al., 2013; Mali et al., 2013a).Meganuclease, ZF, and TALE proteins all recognize specific

DNA sequences through protein-DNA interactions. Althoughmeganucleases integrate its nuclease and DNA-bindingdomains, ZF and TALE proteins consist of individual modulestargeting 3 or 1 nucleotides (nt) of DNA, respectively(Figure 2B). ZFs and TALEs can be assembled in desired combi-nations and attached to the nuclease domain of FokI to directnucleolytic activity toward specific genomic loci. Each of theseplatforms, however, has unique limitations.Meganucleases have not been widely adopted as a genome

engineering platform due to lack of clear correspondencebetween meganuclease protein residues and their target DNAsequence specificity. ZF domains, on the other hand, exhibitcontext-dependent binding preference due to crosstalk betweenadjacent modules when assembled into a larger array (Maederet al., 2008). Although multiple strategies have been developedto account for these limitations (Gonzaelz et al., 2010; Sanderet al., 2011), assembly of functional ZFPs with the desired DNAbinding specificity remains a major challenge that requires anextensive screening process. Similarly, although TALE DNA-binding monomers are for the most part modular, they can stillsuffer from context-dependent specificity (Juillerat et al., 2014),and their repetitive sequences render construction of novelTALE arrays labor intensive and costly.Given the challenges associated with engineering of modular

DNA-binding proteins, new modes of recognition would signifi-cantly simplify the development of custom nucleases. TheCRISPR nuclease Cas9 is targeted by a short guide RNA thatrecognizes the target DNA via Watson-Crick base pairing(Figure 2C). The guide sequence within these CRISPR RNAstypically corresponds to phage sequences, constituting the nat-ural mechanism for CRISPR antiviral defense, but can be easilyreplaced by a sequence of interest to retarget the Cas9nuclease. Multiplexed targeting by Cas9 can now be achievedat unprecedented scale by introducing a battery of short guide

Figure 1. Applications of Genome EngineeringGenetic and epigenetic control of cells with genome engineering technologiesis enabling a broad range of applications from basic biology to biotechnologyand medicine. (Clockwise from top) Causal genetic mutations or epigeneticvariants associated with altered biological function or disease phenotypes cannow be rapidly and efficiently recapitulated in animal or cellular models (Animalmodels, Genetic variation). Manipulating biological circuits could also facilitatethe generation of useful synthetic materials, such as algae-derived, silica-based diatoms for oral drug delivery (Materials). Additionally, precise geneticengineering of important agricultural crops could confer resistance to envi-ronmental deprivation or pathogenic infection, improving food security whileavoiding the introduction of foreign DNA (Food). Sustainable and cost-effec-tive biofuels are attractive sources for renewable energy, which could beachieved by creating efficient metabolic pathways for ethanol production inalgae or corn (Fuel). Direct in vivo correction of genetic or epigenetic defects insomatic tissue would be permanent genetic solutions that address the rootcause of genetically encoded disorders (Gene surgery). Finally, engineeringcells to optimize high yield generation of drug precursors in bacterial factoriescould significantly reduce the cost and accessibility of useful therapeutics(Drug development).

Cell 157, June 5, 2014 ª2014 Elsevier Inc. 1263

Hsu et al., Cell (2014)

Basic research: reverse genetics - probing gene function - loss-of-function or gain-of-function screens

Synthetic biology & biotech - pathway engineering in industrial microbes for making new compounds - engineered agricultural crops for food and biofuel production

Applied research: Animal & cell-based models - specific mutations to mimic human disorders - screening and target validation

Somatic gene therapy - gene correction ex vivo - delivery to specific tissues

CRISPR-Cas and gene therapy

Gene therapy 1.0: Gene augmentation

• Developed in 1990’s before genome editing became possible• Basic idea: Deliver a healthy copy of a gene into the cells (gene augmentation)

• Problems:• Immunogenicity (adenovirus)• Difficult to control site of integration (retroviruses)

Gene therapy 2.0: Genome editing

• Basic idea: Replace a defective gene with a healthy one in the same location by “overwriting” the mutation that causes the disease

CRISPR-Cas9: Proof-of-concept studies in model systems

Gene correction• hereditary tyrosinemia (hydrodynamic injection into mouse liver)• Duchenne muscular dystrophy (mouse model, patient-derived iPSCs)• Beta-thalassemia (iPSC model)• cystic fibrosis (patient-derived iPSCs and intestinal stem cell organoids)• CCR5 (HIV-1 resistance) in CD4 lymphocytes and HSPCs

Targeting viruses• HIV, Hepatitis B, Papillomavirus, Epstein-Barr

Somatic vs. germline genome editing in gene therapy

Somatic• editing is performed in cells and tissues

where the disease manifests itself (e.g. liver, neurons, retina)

• or in stem cells that do not give rise to germline (e.g. hematopoietic stem cells)

• patient does not transmit the modification to the progeny

Germline• editing is performed in germ cells (eggs

or sperm) or germ stem cells that give rise to eggs or sperm

• or in early developing embryo

• all (nearly) tissues in the patient are modified

• the modification can be passed to offspring

Currently permitted but heavily regulated in most jurisdictions

Banned in many jurisdictions and strictly restricted in the rest

Somatic gene therapy: ex-vivo genome editingEx-vivo

Inherited disorders of the blood (e.g. sickle cell anemia, heamophilia, thalassemia)

or the immune system (SCID)

Genome editing in zygotes/embryos

transcribed in developing endosperm (supple-mentary text). Peak transcript abundance coin-cides with the dramatic increase in linoleic acidcontent that occurs during seed development atthe perisperm-endosperm transition (27).Our analysis of the adaptive genomic land-

scape of C. canephora identifies the convergentevolution of caffeine biosynthesis among plantlineages and establishes coffee as a reference spe-cies for understanding the evolution of genomestructure in asterid angiosperms.

REFERENCES AND NOTES

1. E. Robbrecht, J. F. Manen, Syst. Geogr. Plants 76, 85–146 (2006).2. P. Lashermes et al., Mol. Gen. Genet. 261, 259–266 (1999).3. M. Noirot et al., Ann. Bot. (London) 92, 709–714 (2003).4. Materials and methods are available as supplementary

materials on Science Online.5. S. Schaack, C. Gilbert, C. Feschotte, Trends Ecol. Evol. 25,

537–546 (2010).6. A. Roulin et al., BMC Evol. Biol. 9, 58 (2009).7. M. El Baidouri et al., Genome Res. 24, 831–838 (2014).8. C. Moisy, A. H. Schulman, R. Kalendar, J. P. Buchmann,

F. Pelsy, Theor. Appl. Genet. 127, 1223–1235 (2014).9. O. Jaillon et al., Nature 449, 463–467 (2007).10. S. Sato et al., Nature 485, 635–641 (2012).11. P. Librado, F. G. Vieira, J. Rozas, Bioinformatics 28, 279–281 (2012).

12. S. H. Hulbert, C. A. Webb, S. M. Smith, Q. Sun, Annu. Rev.Phytopathol. 39, 285–312 (2001).

13. L. McHale, X. Tan, P. Koehl, R. W. Michelmore, Genome Biol. 7,212 (2006).

14. F. Gleason, R. Chollet, Plant Biochemistry (Jones andBartlett, Sudbury, MA, 2011).

15. J. A. Nathanson, Science 226, 184–187 (1984).16. A. Pacheco, J. Pohlan, M. Schulz, Allelopathy J. 21, 39–56 (2008).17. H. Ashihara, H. Sano, A. Crozier, Phytochemistry 69, 841–856

(2008).18. A. A. McCarthy, J. G. McCarthy, Plant Physiol. 144, 879–889 (2007).19. M. Ogawa, Y. Herai, N. Koizumi, T. Kusano, H. Sano, J. Biol.

Chem. 276, 8213–8218 (2001).20. E. Pichersky, E. Lewinsohn, Annu. Rev. Plant Biol. 62,

549–566 (2011).21. B. Field, A. E. Osbourn, Science 320, 543–547 (2008).22. M. Matsuno et al., Science 325, 1688–1692 (2009).23. B. Field et al., Proc. Natl. Acad. Sci. U.S.A. 108, 16116–16121 (2011).24. J. Zhang, R. Nielsen, Z. Yang, Mol. Biol. Evol. 22, 2472–2479

(2005).25. D. Villarreal et al., J. Agric. Food Chem. 57, 11321–11327 (2009).26. S. Dussert, A. Laffargue, A. de Kochko, T. Joët, Phytochemistry

69, 2950–2960 (2008).27. T. Joët et al., New Phytol. 182, 146–162 (2009).

ACKNOWLEDGMENTS

We acknowledge the following sources for funding: ANR-08-GENM-022-001 (to P.L.); ANR-09-GENM-014-002 (to P.W.); AustralianResearch Council (to R.J.H.); Natural Sciences and EngineeringResearch Council of Canada (to D.S.); CNR-ENEA Agrifood

Project A2 C44 L191 (to G.Gi.); FINEP-Qualicafé, INCT-CAFÉ(to A.C.A.); NSF grants 0922742 (to V.A.A.) and 0922545 (to R.M.);and the College of Arts and Sciences, University at Buffalo(to V.A.A.). We thank P. Facella (ENEA) for Roche 454 sequencingand Instituto Agronômico do Paraná (Paraná, Brazil) for fruitRNA. This work was supported by the high-performance cluster ofthe SouthGreen Bioinformatics platform (UMR AGAP) CIRAD(www.southgreen.fr). The C. canephora genome assembly andgene models are available on the Coffee Genome Hub(http://coffee-genome.org) and the CoGe platform(www.genomevolution.org). Sequencing data are deposited inthe European Nucleotide Archive under the accession numbersCBUE020000001 to CBUE020025216 (contigs), HG739085to HG752429 (scaffolds), and HG974428 to HG974439(chromosomes). Gene family alignments and phylogenetic treesfor BAHD acyltransferases and NMTs are available in theGreenPhylDB (www.greenphyl.org/cgi-bin/index.cgi) under the genefamily IDs CF158535 and CF158539 to CF158545, respectively.We declare no competing financial interests.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/345/6201/1181/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S33Tables S1 to S27References (28–175)

28 April 2014; accepted 29 July 201410.1126/science.1255274

GENOME EDITING

Prevention of muscular dystrophyin mice by CRISPR/Cas9–mediatedediting of germline DNAChengzu Long,1* John R. McAnally,1* John M. Shelton,2 Alex A. Mireault,1

Rhonda Bassel-Duby,1 Eric N. Olson1†

Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutationsin the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD ischaracterized by progressive muscle weakness and a shortened life span, and there is noeffective treatment.We used clustered regularly interspaced short palindromic repeat/Cas9(CRISPR/Cas9)–mediated genome editing to correct the dystrophin gene (Dmd) mutation inthe germ line ofmdxmice, amodel for DMD, and thenmonitoredmuscle structure and function.Genome editing produced genetically mosaic animals containing 2 to 100% correction of theDmd gene.The degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency ofgene correction, likely reflecting an advantage of the corrected cells and their contribution toregenerating muscle.With the anticipated technological advances that will facilitate genomeediting of postnatal somatic cells, this strategymayone day allowcorrection of disease-causingmutations in the muscle tissue of patients with DMD.

Duchenne muscular dystrophy (DMD) iscaused by mutations in the gene for dys-trophin on the X chromosome and affectsapproximately 1 in 3500 boys. Dystrophinis a large cytoskeletal structural protein

essential formuscle cellmembrane integrity.With-out it, muscles degenerate, causing weakness andmyopathy (1). Death of DMD patients usuallyoccurs by age 25, typically from breathing com-plications and cardiomyopathy. Hence, therapyfor DMD necessitates sustained rescue of skele-tal, respiratory, and cardiac muscle structureand function. Although the genetic cause ofDMD was identified nearly three decades ago(2), and several gene- and cell-based therapieshave been developed to deliver functional Dmdalleles or dystrophin-like protein to diseased mus-cle tissue, numerous therapeutic challenges have

been encountered, and no curative treatmentexists (3).RNA-guided, nuclease-mediated genome edit-

ing, based on type II CRISPR (clustered regu-larly interspaced short palindromic repeat)/Cas(CRISPR-associated) systems, offers a new ap-proach to alter the genome (4–6). In brief, Cas9,a nuclease guided by single-guide RNA (sgRNA),binds to a targeted genomic locus next to theprotospacer adjacent motif (PAM) and generatesa double-strand break (DSB). The DSB is thenrepaired either by nonhomologous end-joining(NHEJ), which leads to insertion/deletion (indel)mutations, or by homology-directed repair (HDR),which requires an exogenous template and cangenerate a precise modification at a target locus(7). Unlike other gene therapy methods, whichadd a functional, or partially functional, copy of agene to a patient’s cells but retain the originaldysfunctional copy of the gene, this system canremove the defect. Genetic correction using en-gineered nucleases (8–12) has been demonstratedin immortalized myoblasts derived from DMDpatients in vitro (9), and rodent models of rarediseases (13), but not yet in animal models ofrelatively common and currently incurable dis-eases, such as DMD.The objective of this study was to correct the

genetic defect in the Dmd gene of mdx mice byCRISPR/Cas9–mediated genome editing in vivo.Themdxmouse (C57BL/10ScSn-Dmdmdx/J) con-tains a nonsensemutation in exon 23 of theDmdgene (14, 15) (Fig. 1A). We injected Cas9, sgRNA,and HDR template intomouse zygotes to correctthe disease-causing gene mutation in the germline (16, 17), a strategy thathas thepotential to correctthemutation in all cells of the body, including myo-genic progenitors. Safety and efficacy of CRISPR/Cas9–based gene therapy was also evaluated.

1184 5 SEPTEMBER 2014 • VOL 345 ISSUE 6201 sciencemag.org SCIENCE

1Department of Molecular Biology and Hamon Center forRegenerative Science and Medicine, University of TexasSouthwestern Medical Center, Dallas, TX 75390, USA.2Department of Internal Medicine, University of TexasSouthwestern Medical Center, Dallas, TX 75390, USA.*These authors contributed equally to this work. †To whomcorrespondence should be addressed. E-mail: eric.olson@utsouthwestern.edu

RESEARCH | REPORTS

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Initially, we tested the feasibility and opti-mized the conditions of CRISPR/Cas9–mediatedDmd gene editing inwild-typemice (C57BL6/C3Hand C57BL/6) (see the supplementary materials).We designed a sgRNA to target Dmd exon 23(fig. S1A) and a single-stranded oligodeoxynucleo-tide (ssODN) as a template for HDR-mediatedgene repair (fig. S1B and table S1). The wild-type zygotes were co-injected with Cas9 mRNA,sgRNA-DMD, and ssODN and then implantedinto pseudopregnant female mice. Polymerasechain reaction (PCR) products corresponding toDmd exon 23 from progeny mice were sequenced(fig. S1, C to E). Efficiency of CRISPR/Cas9–mediatedDmd gene editing is shown in table S2.We next applied the optimized CRISPR/Cas9–

mediated genomic editing method to mdx mice(Fig. 1B). The CRISPR/Cas9–mediated genomicediting system will correct the point mutation inmdx mice during embryonic development viaHDR or NHEJ (Fig. 1, C and D, and fig. S2A).“Corrected” mdx progeny (termed mdx-C) wereidentified by restriction fragment length poly-morphism (RFLP) analysis and the mismatch-specific T7 endonuclease I (T7E1) assay (Fig. 1E,table S2, and supplementary materials). We an-alyzed a total of 11 differentmdx-C mice. PCR pro-ducts of Dmd exon 23 from seven mdx-C micewithHDR-mediated gene correction (termedmdx-C1 to C7) and four mdx-C mice containing NHEJ-mediated in-frame deletions of the stop codon

(termed mdx-N1 to N4) were sequenced. Se-quencing results revealed that CRISPR/Cas9–mediated germline editing produced geneticallymosaic mdx-C mice displaying from 2 to 100%correction of the Dmd gene (Fig. 1E and fig. S2,B and C). A wide range of mosaicism occurs ifCRISPR/Cas9–mediated repair occurs after thezygote stage, resulting in genomic editing in asubset of embryonic cells (18). All mouse progenydeveloped to adultswithout signs of tumor growthor other abnormal phenotypes.We tested four different mouse groups for pos-

sible off-target effects of CRISPR/Cas9–mediatedgenome editing: (A) mdx mice without treat-ment (termedmdx), (B) CRISPR/Cas9–editedmdxmice (termedmdx+Cas9), (C) Wild-type controlmice (C57BL6/C3H) without treatment (termedWT) and (D) CRISPR/Cas9–edited wild-type mice(termed WT+Cas9) (fig. S3A). Sequences of thetarget site (Dmd exon 23) and a total of 32 po-tential off-target (OT) sites in the mouse ge-nome were predicted by the CRISPR design tool(http://crispr.mit.edu) and are listed in table S3.Ten of the 32 sites, termed OT-01 through OT-10,represent the genome-wide “top-10 hits.” Twenty-two of the 32, termed OTE-01 through OTE-22are located within exons.Deep sequencing of PCR products correspond-

ing to Dmd exon 23 revealed high ratios of HDRandNHEJ-mediated geneticmodification in groupsB and D but not in control groups A and C (fig.

S3A and table S4). There was no difference in thefrequency of indel mutations in the 32 potentialoff-target regions among the different groups (fig.S3, B and C, table S5, and supplementary mate-rials). These results are also consistent with recentgenome-wide studies showing that DNA cleavageby Cas9 is not promiscuous (19–21). Thus, off-target effects may be less of a concern in vivothan previously observed in vitro (22, 23).To analyze the effect of CRISPR/Cas9–mediated

genomic editing on the development of musculardystrophy, we performed histological analyses offour different muscle types [quadriceps, soleus(hindlimbmuscle), diaphragm(respiratorymuscle),and heart muscle] from wild-type mice, mdxmice, and three chosen mdx-C mice with dif-ferent percentages of Dmd gene correction at 7to 9 weeks of age. mdx muscle showed histo-pathologic hallmarks of muscular dystrophy, in-cluding variation in fiber diameter, centralizednuclei, degenerating fibers, necrotic fibers, andmineralized fibers, as well as interstitial fibrosis(Fig. 2 and figs. S4A and S5A). Immunohisto-chemistry showed no dystrophin expression inskeletal muscle or heart of mdx mice, whereaswild-type mice showed dystrophin expressionin the subsarcolemmal region of the fibers andthe heart (Fig. 2). Although mdx mice carry astop mutation in the Dmd gene, we observed0.2 to 0.6% revertant fibers, consistent with aprevious report (24).mdx-Cmice with 41% of the

SCIENCE sciencemag.org 5 SEPTEMBER 2014 • VOL 345 ISSUE 6201 1185

Fig. 1. CRISPR/Cas9–mediated Dmd correction inmdxmice. (A) Schematicof the targeted exon ofmouseDmd and sequence fromwild-type (upper) andmdxmice (lower).Themdx pointmutation (C to T) ismarked in red, and the prematurestop codon is underlined. (B) Schematic of the 20-nucleotide sgRNA target se-quenceof themdxallele (blue) and thePAM(red).The red arrowhead indicates theCas9 cleavage site. ssODN, which contains 90 base pairs (bp) of homologysequence flanking each side of the target site, was used as HDR template. ssODNincorporates four silent mutations (green) and adds a TseI restriction enzyme site(underlined) for genotyping and quantification of HDR-mediated gene editing (fig.S1B). (C) Schematic for the gene correction by HDR or NHEJ.The correspondingDNA and protein sequences are shown in fig. S2A. (D) Strategy of the gene cor-

rection in mdx mice via germline gene therapy. (E) Genotyping results of mdx-Cmice withmosaicism of 2 to 100%correctedDmd gene. Undigested PCRproduct(upper panel),TseI digestion (middle panel), and T7E1 digestion (lower panel) on a2% agarose gel. The red arrowhead in the middle panel marks the DNA bandindicating HDR-mediated correction generated by TseI digestion. The blue arrow-headmarks the DNA band of the uncorrectedmdx allele.The relative intensity oftheDNAbands (indicated by blue and red arrowheads) reflects the percentage ofHDR in the genomic DNA. The percentage of HDR is located under the middlepanel. The band intensity was quantified by ImageJ (NIH). The blue and redarrowheads in the lower panel indicate uncut and cut bands byT7E1, respectively.M denotes size marker lane. bp indicates the length of the marker bands.

RESEARCH | REPORTS

• pronuclear injection of Cas9 mRNA and sgRNA• HDR repair using oligonucleotide template

Long et al., Science (2014)

Germline genome editing in humans?Feature p. 48

HP Tries to Reinvent the Computer

Business Report p. 63

Persuasion

Review p. 72

The Problem with Fake Meat

VOL. 118 NO. 3 MAY/JUNE 2015 $6.99

p26

WE CANNOW

ENGINEERTHE

HUMAN RACE

Genome editing in human embryos

RESEARCH ARTICLE

CRISPR/Cas9-mediated gene editing in humantripronuclear zygotes

Puping Liang, Yanwen Xu, Xiya Zhang, Chenhui Ding, Rui Huang, Zhen Zhang, Jie Lv, Xiaowei Xie,Yuxi Chen, Yujing Li, Ying Sun, Yaofu Bai, Zhou Songyang, Wenbin Ma, Canquan Zhou&, Junjiu Huang&

Guangdong Province Key Laboratory of Reproductive Medicine, the First Affiliated Hospital, and Key Laboratory of GeneEngineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China& Correspondence: hjunjiu@mail.sysu.edu.cn (J. Huang), zhoucanquan@hotmail.com (C. Zhou)Received March 30, 2015 Accepted April 1, 2015

ABSTRACT

Genome editing tools such as the clustered regularlyinterspaced short palindromic repeat (CRISPR)-associ-ated system (Cas) have been widely used to modifygenes in model systems including animal zygotes andhuman cells, and hold tremendous promise for bothbasic research and clinical applications. To date, a se-rious knowledge gap remains in our understanding ofDNA repair mechanisms in human early embryos, and inthe efficiency and potential off-target effects of usingtechnologies such as CRISPR/Cas9 in human pre-im-plantation embryos. In this report, we used tripronuclear(3PN) zygotes to further investigate CRISPR/Cas9-me-diated gene editing in human cells. We found thatCRISPR/Cas9 could effectively cleave the endogenousβ-globin gene (HBB). However, the efficiency of ho-mologous recombination directed repair (HDR) of HBBwas low and the edited embryos were mosaic. Off-targetcleavage was also apparent in these 3PN zygotes asrevealed by the T7E1 assay and whole-exome se-quencing. Furthermore, the endogenous delta-globingene (HBD), which is homologous to HBB, competedwith exogenous donor oligos to act as the repair tem-plate, leading to untoward mutations. Our data alsoindicated that repair of the HBB locus in these embryosoccurred preferentially through the non-crossover HDRpathway. Taken together, our work highlights the

pressing need to further improve the fidelity and speci-ficity of the CRISPR/Cas9 platform, a prerequisite forany clinical applications of CRSIPR/Cas9-mediatedediting.

KEYWORDS CRISPR/Cas9, β-thalassemia, humantripronuclear zygotes, gene editing, homologousrecombination, whole-exome sequencing

INTRODUCTION

The CRISPR/Cas9 RNA-endonuclease complex, consistingof the Cas9 protein and the guide RNA (gRNA) (∼99 nt), isbased on the adaptive immune system of streptococcuspyogenes SF370. It targets genomic sequences containingthe tri-nucleotide protospacer adjacent motif (PAM) andcomplementary to the gRNA, and can be programmed torecognize virtually any genes through the manipulation ofgRNA sequences (Cho et al., 2013; Cong et al., 2013; Jineket al., 2012; Jinek et al., 2013; Mali et al., 2013c). FollowingCas9 binding and subsequence target site cleavage, thedouble strand breaks (DSBs) generated are repaired by ei-ther non-homologous end joining (NHEJ) or homologousrecombination directed repair (HDR), resulting in indels orprecise repair respectively (Jinek et al., 2012; Moynahan andJasin, 2010). The ease, expedience, and efficiency of theCRISPR/Cas9 system have lent itself to a variety of appli-cations, including genome editing, gene function investiga-tion, and gene therapy in animals and human cells (Changet al., 2013; Cho et al., 2013; Cong et al., 2013; Friedlandet al., 2013; Hsu et al., 2014; Hwang et al., 2013; Ikmi et al.,2014; Irion et al., 2014; Jinek et al., 2013; Li et al., 2013a; Liet al., 2013b; Long et al., 2014; Ma et al., 2014; Mali et al.,2013c; Niu et al., 2014; Smith et al., 2014a; Wu et al., 2013;Wu et al., 2014b; Yang et al., 2013).

Puping Liang, Yanwen Xu, Xiya Zhang and Chenhui Ding havecontributed equally to this work.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s13238-015-0153-5) contains supplementarymaterial, which is available to authorized users.

© The Author(s) 2015. This article is published with open access at Springerlink.com and journal.hep.com.cn

Protein CellDOI 10.1007/s13238-015-0153-5 Protein&Cell

Protein

&Cell

• used non-viable embryos (derived from tripronuclear zygotes)• attempted to edit the beta-globin gene involved in beta-thalassemia• efficiency of homology-directed repair was low (4 out of 86)• embryos were mosaic (mixture of modified and unmodified cells)• authors concluded that clinical applications would be premature

The ethical aspect of human germline genome modification

36 3 APRIL 2015 • VOL 348 ISSUE 6230 sciencemag.org SCIENCE

PERSPECTIVES

Genome engineering technology offers

unparalleled potential for modifying

human and nonhuman genomes. In

humans, it holds the promise of cur-

ing genetic disease, while in other

organisms it provides methods to

reshape the biosphere for the benefit of the

environment and human societies. However,

with such enormous opportunities come un-

known risks to human health

and well-being. In January, a

group of interested stakehold-

ers met in Napa, California ( 1), to discuss the

scientific, medical, legal, and ethical impli-

cations of these new prospects for genome

biology. The goal was to initiate an informed

discussion of the uses of genome engineer-

ing technology, and to identify those areas

where action is essential to prepare for fu-

ture developments. The meeting identified

immediate steps to take toward ensuring

that the application of genome engineering

technology is performed safely and ethically.

The promise of so-called “precision

medicine” is propelled in part by syner-

gies between two powerful technologies:

DNA sequencing and genome engineering.

Advances in DNA sequencing capabilities

and genome-wide association studies have

provided critical information about the ge-

netic changes that influence the develop-

ment of disease. In the past, without the

means to make specific and efficient modi-

fications to a genome, the ability to act on

this information was limited. However, this

limitation has been upended by the rapid

development and widespread adoption of a

simple, inexpensive, and remarkably effec-

tive genome engineering method known as

clustered regularly interspaced short palin-

dromic repeats (CRISPR)–Cas9 ( 2). Build-

ing on predecessor platforms, a rapidly

expanding family of CRISPR-Cas9–derived

technologies is revolutionizing the fields of

genetics and molecular biology as research-

ers employ these methods to change DNA

sequences—by introducing or correcting

genetic mutations—in a wide variety of cells

and organisms.

CURRENT APPLICATIONS. The simplicity

of the CRISPR-Cas9 system allows any re-

searcher with knowledge of molecular bi-

ology to modify genomes, making feasible

experiments that were previously difficult

or impossible to conduct. For example, the

CRISPR-Cas9 system enables introduc-

tion of DNA sequence changes that cor-

rect genetic defects in whole animals, such

as replacing a mutated gene underlying

liver-based metabolic disease in a mouse

model ( 3). The technique also allows DNA

sequence changes in pluripotent embryonic

stem cells ( 4) that can then be cultured to

produce specific tissues, such as cardiomyo-

cytes or neurons ( 5). Such studies are lay-

ing the groundwork for refined approaches

that could eventually treat human disease.

CRISPR-Cas9 technology can also be used

to replicate precisely the genetic basis for

human diseases in model organisms, lead-

ing to unprecedented insights into previ-

ously enigmatic disorders.

In addition to facilitating changes in dif-

ferentiated somatic cells of animals and

plants, CRISPR-Cas9 technology, as well

as other genome engineering methods, can

be used to change the DNA in the nuclei of

reproductive cells that transmit informa-

tion from one generation to the next (an

A prudent path forward for genomic

engineering and germline gene modification

By David Baltimore ,1 Paul Berg, 2

Michael Botchan ,3, 4 Dana Carroll, 5

R. Alta Charo, 6 George Church, 7

Jacob E. Corn, 4 George Q. Daley ,8, 9

Jennifer A. Doudna ,4, 10 * Marsha Fenner ,4

Henry T. Greely, 11 Martin Jinek, 12

G. Steven Martin, 13 Edward Penhoet, 14

Jennifer Puck, 15 Samuel H. Sternberg ,16

Jonathan S. Weissman ,4, 17

Keith R. Yamamoto4, 18

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the

human genome is urgently needed

BIOTECHNOLOGY

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It is thought that studies involving the use of genome-editing tools to modify the DNA of human embryos will be

published shortly1. There are grave concerns regarding

the ethical and safety implications of this research. There is also fear of the negative impact it could have on important work involving the use of genome-editing tech-niques in somatic (non-reproductive) cells.

We are all involved in this latter area of work. One of us (F.U.) helped to develop the first genome-editing technology, zinc-finger nucleases2 (ZFNs), and is now senior scientist at the company developing them, Sangamo BioSciences of Richmond, California. The Alliance for Regenerative Medicine (ARM; in which E.L., M.W. and S.E.H. are involved), is an international organization that represents more than 200 life-sciences companies, research institutions, non-profit organizations, patient-advocacy groups and investors focused on developing and com-mercializing therapeutics, including those involving genome editing.

Genome-editing technologies may offer a powerful approach to treat many human diseases, including HIV/AIDS, haemo-philia, sickle-cell anaemia and several forms of cancer3. All techniques currently in various stages of clinical development focus on modifying the genetic material of somatic cells, such as T cells (a type of white blood cell). These are not designed to affect sperm or eggs.

In our view, genome editing in human embryos using current technologies could have unpredictable effects on future gen-erations. This makes it dangerous and ethi-cally unacceptable. Such research could be exploited for non-therapeutic modifica-tions. We are concerned that a public outcry about such an ethical breach could hinder a promising area of therapeutic development, namely making genetic changes that cannot be inherited.

At this early stage, scientists should agree not to modify the DNA of human repro-ductive cells. Should a truly compelling case ever arise for the therapeutic benefit

of germ line modification, we encourage an open discussion around the appropriate course of action.

EDITING TOOLSGenome editing of human somatic cells aims to repair or eliminate a mutation that could cause disease. The premise is that corrective changes to a sufficient number of cells carrying the mutation — in which the genetic fixes would last the lifetimes of the modified cells and their progeny — could provide a ‘one and done’ curative treatment for patients.

For instance, ZFNs are DNA-binding proteins that can be engineered to induce a double-strand break in a section of DNA. Such molecular scissors enable researchers to ‘knock out’ specific genes, repair a muta-tion or incorporate a new stretch of DNA into a selected location.

Sangamo BioSciences is conducting clinical trials to evaluate an application of genome editing as a potential ‘functional cure’ for HIV/AIDS4. The hope is that

Don’t edit the human germ line

Heritable human genetic modifications pose serious risks, and the therapeutic benefits are tenuous, warn Edward Lanphier, Fyodor Urnov and colleagues.

SHU

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COMMENT

© 2015 Macmillan Publishers Limited. All rights reserved

Recommendations1. Strongly discourage germline genome modification for clinical applications for the time being 2. Create forums for information and educating about potential rewards and risks of genome

editing3. Encourage support of transparent research to evaluate specificity and efficacy 4. Convene a globally representative group of researchers, bioethicists, government agencies

and public bodies to recommend policies

Legal framework for human genome modification

Legal framework for human genome modification

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Taiyuan

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Wuhan

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Cartagena

Cúcuta

MedellínAbidjan

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Santiago

Guayaquil

Alexandria

Aswan

Bordeaux

Lille

Lyon

MarseilleToulouse

Cologne

Frankfurt

Hamburg

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Qaanaaq (Thule)

Agra

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Chennai

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Patna

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Bandung

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Malang

Medan

Padang

Palembang

Pekanbaru

Pontianak Samarinda

Semarang Surabaya

Tanjungkarang-Telukbetung

Ahvaz

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Mashhad

Qom

Zahedan

Al Basrah

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Mosul

Genoa

Milan

Naples

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HiroshimaNagoya

Osaka

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Kashi

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Atyraü

Qaraghandy(Karaganda)

Shymkent

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Blantyre

TimbuktuAcapulco

Cancún

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CiudadJuarez

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Matamoros

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Iquitos

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Kraków

Porto

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Anadyr'Arkhangel'sk

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KharkivL'viv

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QUEEN ELIZABETH

ISLANDS

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NEW SIBERIAN ISLANDS

SEVERNAYAZEMLYA

FRANZ JOSEFLAND

NOVAYAZEMLYA

EllesmereIsland

BanksIsland

VictoriaBaffinIsland

Island

Rockall

SakhalinIsland ofNewfoundland

Sardinia

Sicily

Crete

Minami-tori-shima

HainanDao

Socotra

PENEDOS DESÃO PEDRO E SÃO PAULO

Kiritimati(Christmas Island)

Annobon

DiegoGarcia

Martin Vaz

Trindade

Isla San AmbrosioEaster Island

Lord HoweIsland

Île Amsterdam

Île Saint-Paul

Tasmania

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GALAPAGOSISLANDS

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ÎLES MARQUISES

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ÎLES TUBUAI

ARCHIPEL DES TUAMOTU

ARCHIPIÉLAGOJUAN FERNÁNDEZ

SOUTH ORKNEYISLANDS

PRINCE EDWARDISLANDS

ÎLES CROZET

ÎLES KERGUELEN

CHATHAM ISLANDS

BOUNTY ISLANDSSNARES ISLANDS

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ARQUIPÉLAGO DEFERNANDO DE NORONHA

Isla San Felíx

N A M

P O - S H

O T O

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A L E U T I A N I S L A N D S

803622AI (G00010) 8-13

Bermuda

AUSTRALIA

Sicily / AZORES

Dependency or area of special sovereignty

Capital

Scale 1:35,000,000

standard parallels 38°N and 38°SRobinson Projection

Island / island group

Independent state

Political Map of the World, August 2013

August 2013Boundary representation is not necessarily authoritative.

UNITED STATES• NIH does not accept clinical trial proposals

involving germline genome modification • Federal ban on funding research using

human embryos• Privately-funded research is not regulated

CHINA• Ban on using genetically modified germ cells or

embryos for reproductive purposes• Genetic modifications for research are permitted

GERMANY• Strict legal ban on genetic

modification of germ cells (also for research) and their use for reproductive purposes

SWITZERLAND• Constitutional ban on cloning and

other manipulation of the genetic material in eggs, sperm or embryos

UNITED KINGDOM• Strict ban on using genetically modified germ cells

or embryos for reproductive purposes• Regulators may permit germline modification for

research• Currently, an application is pending for research

license from HFEA (Kathy Niakan)

The ethical controversy of human germline genome modification

Even if it can be made 100% safe, should it go ahead?Is there actually need for therapeutic germline genome editing?

What about non-therapeutic use?

Is it efficient and safe enough to go ahead now?

Human improvement by genome editing

Naturally occurring protective or beneficial gene variants

LRP5 G171V / – extra-strong bones, prevents osteoporosis

MSTN (myostatin) – / – lean and hypertrophied muscles

SCN9A – / – insensitivity to pain

ABCC11 – / – low body odour production

CCR5 – / – or Δ32/Δ32 HIV resistance

PCSK9 – / – low incidence of coronary disease

APP (amyloid precursor protein)

A673T / – low incidence of Alzheimer’s disease

GHR – / – low incidence of cancer

SLC30A8 – / + low type II diabetes

IFIH1 E627X/ + low type I diabetes

The need for a global debate

“Human gene editing offers great promise for improving human health and well-being but it also raises significant ethical and societal issues. It is vital that we have a well-informed international debate about the potential benefits and risks, and this summit can hopefully set the tone for that discussion.”

Sir Paul Nurse, President of the Royal Society

1

International Summit on Human Gene Editing: A Global Discussion

Convened by:

Chinese Academy of Sciences

The Royal Society U.S. National Academy of Sciences U.S. National Academy of Medicine

National Academy of Sciences Building

2101 Constitution Avenue, N.W. Washington, D.C. 20418

December 1-3, 2015

Agenda

Tuesday, 1 December 2015

7:00 am Registration/Continental Breakfast

8:00 am Welcome and Introductions

Summit Committee Chair David Baltimore, California Institute of Technology

8:05 am Opening Remarks

Congressman Bill Foster (D-11th District, Illinois) John P. Holdren, White House Office of Science and Technology Policy Representatives of the Convening Organizations:

Ralph J. Cicerone, U.S. National Academy of Sciences Victor J. Dzau, U.S. National Academy of Medicine Zhihong Xu, Chinese Academy of Sciences John Skehel, The Royal Society

Conclusions: the promise and risks of the CRISPR revolution

• CRISPR-Cas9 is a powerful genome engineering technology

• Transformative capabilities in basic research, biotech and biomedicine

• Proof-of-concept studies show great promise for somatic gene therapy

• So far, no clinical trials yet, but very likely in the next few years

• The application of CRISPR-Cas9 germline genome modification in humans is highly controversial

• There is need for a global discussion and consensus on the use