from/atInstBiophys,CASonJune29,2014
Minireviews:ACutabovetheRest:TargetedGenomeEditingTechnologiesinHumanPluripotentStemCells
MoLi,KeiichiroSuzuki,NaYoungKim,Guang-HuiLiuandJuanCarlosIzpisuaBelmonte
J.Biol.Chem.2014,289:4594-4599.doi:10.1074/jbc.R113.488247originallypublishedonlineDecember20,2013 essthemostupdatedversionofthisarticleatdoi:10.1074/jbc.R113.488247 Findarticles,minireviews,ReflectionsandClassicsonicsontheJBCAffinitySites. Alerts:•Whenthisarticleiscited•Whenacorrectionforthisarticleisposted ClickheretochoosefromallofJBC'se-mailalerts Thisarticlecites61references,22ofwhichcanbeessedfreeat#ref-list-
1 MINIREVIEW THEJOURNALOFBIOLOGICALCHEMISTRYVOL.289,NO.8,pp.4594–4599,February21,2014©2014byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PublishedintheU.S.A. Downloadedfrom/atInstBiophys,CASonJune29,2014 ACutabovetheRest:TargetedGenomeEditingTechnologiesinHumanPluripotentStemCells* Published,JBCPapersinPress,December20,2013,DOI10.1074/jbc.R113.488247 MoLi‡1,
2,KeiichiroSuzuki‡1,
2,NaYoungKim‡,Guang-HuiLiu§3,andJuanCarlosIzpisuaBelmonte‡4Fromthe‡GeneExpressionLaboratory,SalkInstituteforBiologicalStudies,LaJolla,California92037andthe§NationalLaboratoryofBiomacromolecules,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,China Humanpluripotentstemcells(hPSCs)offerunprecedentedopportunitiestostudycellulardifferentiationandmodelhumandiseases.TheabilitytopreciselymodifyanygenomicsequenceholdsthekeytorealizingthefullpotentialofhPSCs.ThankstotherapiddevelopmentofnovelgenomeeditingtechnologiesdrivenbytheenormousinterestinthehPSCfield,genomeeditinginhPSCshasevolvedfrombeingadauntingtaskafewyearsagotoaroutineprocedureinmostlaboratories.Here,weprovideanoverviewofthemainstreamgenomeeditingtools,includingzincfingernucleases,transcriptionactivator-likeeffectornucleases,clusteredregularlyinterspacedshortpalindromicrepeat/CAS9RNA-guidednucleases,andhelper-dependentadenoviralvectors.Wediscussthefeaturesandlimitationsofthesetechnologies,aswellashowthesefactorsinfluencetheutilityofthesetoolsinbasicresearchandtherapies. Thediscoveryofinducedpluripotentstemcells(iPSCs)5sevenyearsagohasreignitedtheenthusiasmforcell-basedtherapy.TheabilityofiPSCstoundergounlimiteddivisionwhilemaintaininggenomicintegrityprovidesawaytoethesenescencebarrierofagedsomaticcells.ThecapacityofiPSCstodifferentiateintocellsofthethreegermlayershasbeenextensivelydocumentedinthefield.Takentogether,itisnothardtoappreciatewhyhumaniPSC(hiPSC)-basedautolo- *ThisworkwassupportedinpartbygrantsfromtheG.HaroldandLeilaY. MathersCharitableFoundation,theEllisonMedicalFoundation,theLeonaM.andHarryB.HelmsleyCharitableTrust,andtheGlennFoundationforMedicalResearch(toJ.C.I.B.).ThisisthesixtharticleintheThematicMinireviewSeries“DevelopmentofHumanTherapeuticsBasedonInducedPluripotentStemCell(iPSC)Technology.”1Bothauthorscontributedequallytothiswork.2SupportedbyfellowshipsfromtheCaliforniaInstituteofRegenerativeMedicine.3Supportedbythe“ThousandYoungTalents”programofChina,theNationalLaboratoryofBiomacromolecules,andtheStrategicPriorityResearchProgramoftheChineseAcademyofSciences.4Towhomcorrespondenceshouldbeaddressed.E-mail:belmonte@salk.edu.5Theabbreviationsusedare:iPSC,inducedpluripotentstemcell;hiPSC,humaniPSC;HR,homologousbination;hESC,humanembryonicstemcell;DSB,DNAdouble-strandbreak;NHEJ,non-homologousendjoining;HDR,homology-directedrepair;ZFN,zincfingernuclease;TALEN,transcriptionactivator-likeeffectornuclease;TALE,transcriptionactivatorlikeeffector;CRISPR,clusteredregularlyinterspacedshortpalindromicrepeat;crRNA,CRISPRRNA;tracrRNA,transactivatedcrRNA;HDAdV,helper-dependentadenoviralvector. goustransplantationisheraldedasthefutureofregenerativemedicine(Fig.1).Oneareathathasdrawngreatinterestiscorrectionoficdiseasesinpatient-specifichiPSCswithaprospectofpersonalizedcelltherapy. Pluripotentstemcellsareespeciallyamenableforgenomeeditingbecausetheycanundergoextensivetissueculturemanipulations,suchasdrugselectionandclonalexpansion,whilestillmaintainingtheirpluripotencyandgenomestability.Genetargetinginmouseembryonicstemcellsbyhomologousbination(HR)hasproventobeastapletechniqueforstudyinggenefunction(1,2).However,thesamestrategydoesnottranslatewellintohumanembryonicstemcells(hESCs)orhiPSCs
(3).AlthoughtheclassicalHRmethodhasbeenessfullyusedtogenerateknock-inreporterlinesandtocorrectgenemutationsinhESCs,thereportedtargetingefficienciesareatleastseveralordersofmagnitudelowerthanwhatisachievableinmouseembryonicstemcells(4,5).ThisislikelyduetotheintrinsicdifferencesintheDNArepairprocessbetweenhumansandmice,asmeasurestoimprovesingle-cellsurvivalandDNAtransfectiondidnothaveadramaticeffectongenetargetingefficiency(6,7).However,othermethodsaimedatpromotingHRprovedmorefruitful
(3).Inthepastseveralyears,therehasbeenaspikeofinterestingenomeeditinginhESCsandhiPSCs,possiblyduetothepotentialofthistechnologyinmodelingandcorrectingamyriadoficdiseases(8,9).Thishasfueledarapiddevelopmentinnoveltechnologiesfortargetedmodificationofthehumangenome.Here,weaimtoprovideatimelyupdateonthecurrentgenomeeditingtechnologiesanddiscussthefactorsthatinfluencethechoiceofanappropriatetechnology. GenomeEditingwithSyntheticNucleases IntroductionofaDNAdouble-strandbreak(DSB)triggersDNArepairresponsesviatwomajorpathways(10).TheDSBsarerepairedprimarilybythenon-homologousendjoining(NHEJ)pathway,whicheitherrestorestheoriginalsequenceorcreatessmallinsertionsordeletions(indels).Alternatively,theDSBsmayberepairedthroughHRbasedonahomologousrepairtemplate(aprocesstermedhomology-directedrepair(HDR)).HDRcanbeco-optedtointroducedesiredsequencechangeswhenanexogenoustemplateisintroduced.Therehavebeenseveralapproachesofengineeringsyntheticnucleasestoachievetargetedcleavageofaspecificsiteinthehumangenome.Thesenucleasessharetwoimportantproperties:1)amechanismofrecognizingasufficientlylongtargetsequencethatursonlyonceinthegenomeand2)acatalyticactivitythatisactivatedbysequence-specificbinding. ZincFingerNucleases—Zincfingernucleases(ZFNs)aremodularproteinsconsistingofaseriesoftheCys2-His2zincfingerDNA-bindingmotifsandtheDNAcleavagedomainofthetypeIISrestrictionenzymeFokI(11).Eachzincfingermotifrecognizes3–4bpofDNA,andthemodularnatureofthezincfingermotifallowsspecificbindingtopositesequencebylinkingseveralmotifsintandem.TheactivityoftheFokInucleaserequiresdimerization.Therefore,twoZFNsare 4594JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER8•FEBRUARY21,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs Downloadedfrom/atInstBiophys,CASonJune29,2014 FIGURE1.ConceptualschematicshowingtheapplicationofgenomeeditingandiPSCtechnologiesinregenerativemedicine. designedtobindonoppositesidesofthetargetsitewiththerespectiveFokIdomainsorientedtowardeachother(Fig.1).ThisdesignfurtherenhancesthespecificityofZFNs,asdimerizationofFokIandthuscleavagearedependentonalongertarget.ApairofZFNsthateachcontainsthreezincfingermotifsaresufficienttoensureauniqueintendedtargetofan18-bpsequencebychanceinthehumangenome,althoughfourtosixmotifsaremonlyusedtoincreasespecificity(12).ZFNshavebeenessfullyusedtoeditthegenomeofanisms,includinghumans(11,13). Amongthesyntheticnucleases,ZFNshavetheadvantageofbeingthemoststudied.TherearemanytechnicalresourcestoaidthedesignandassemblyofZFNs.WelldesignedZFNsarehighlyeffectiveinthedisruption,addition,orcorrectionofthetargetgene.Recently,ZFN-mediateddisruptionoftheTAP2geneinhiPSCsmadeitpossibletoproduceanunlimitedamountofantigen-presentingcellsforinationtherapy(14).ZFN-mediatedinsertionoftransgenesintothegenomicsafeharborlocus(AAVS1)ofhiPSCshasbeenusedtoengineercellsforinvivoimagingandtocorrecttheglobinimbalancein␣-thalassemia(15,16).InthecaseofHDR,ZFNsgreatlysimplifytheexperimentaldesign,asshortoligonucleotidesmaybeusedastemplates,andantibioticselectionmaybeomitted(17). ForadetailedountofgenomeeditingofhESCsandhiPSCsusingZFNs,wewouldliketoreferthereaderstorecentreviews(18,19). DespitemanyessfulreportsofZFN-mediatedgenomeeditingofhumancellsinacademicandclinicalresearch,ZFNtechnologyhasseverallimitations.Theassemblyofzincfingermotifsisnotmodularinthestrictestsense.Thebindingaffinityofindividualmotifsiscontext-dependent.Inotherwords,anassembledZFNdoesnotnecessarilyhavehighaffinityforthesequencethatispositeofthe3-bpcognatesequenceofeachzincfingermotif(20).Otherselection-basedmethodshavebeeninventedtoaddressthehighfailurerateofmodularlyassembledZFNs(reviewedinRef.14).Withthesemethods,thenumberoftargetablesitesisreduced.Inanycase,itrequiresaconsiderableamountofexperience,time,andefforttoachieveproficiencyatmakingfunctionalZFNs. Unintendedcleavageatso-calledoff-targetsitesisanotherconcernwithZFNs(21).Off-targetcleavagecouldcausecytotoxicity,introduceunknownmutations,andconfoundtheanalysisoftheeffectsoftheintendedicchanges.FokIvariantsthatformobligateheterodimershavebeenusedtominimizeoff-targetcuttingduetohomodimerizationofZFNs(22).ConvertingFokIintoaDNA-nickingenzymealsohelpsto FEBRUARY21,2014•VOLUME289•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY4595 Downloadedfrom/atInstBiophys,CASonJune29,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs reducetheriskofmutagenesisduetooff-targetcleavagebecausesingle-strandbreaksstimulateHDRontargetwhileminimizingNHEJofftarget(23,24).Evenwiththesetechnicalimprovements,itisstillimportanttomonitoroff-targetcleavageofZFNsintendedfortherapeutics.Bioinformaticstoolsmaybeusedtopredictpotentialoff-targetsites.However,thisapproachdoesnottakeintoconsiderationthedifferencesbetweenthetargetgenomeandthereferencegenome.Furthermore,twostudieshaveshownthattheinvivooff-targetsitesofZFNcleavagecouldnotbefullypredictedbyinvitroorinsilicoanalysis(21,26).Atthecellularlevel,although␥H2AXandp53BP1fociareusedtomonitorDSBsitesaftertheintroductionofZFNs,theycannotdistinguishZFN-mediatedDSBsandspontaneousDSBs.Amoredirectapproachwillbesequencingthegenome,whichisingmorepracticalasnextgenerationsequencingesmoreaffordable.Encouragingly,Yusaetal.(27)foundnoevidenceofoff-targetcleavage-inducedmutationinaZFN-modifiedhiPSClinebyexomesequencing. TranscriptionActivator-likeEffectorNucleases—Recently,anothersyntheticnucleasetermedtranscriptionactivator-likeeffectornuclease(TALEN)hasemergedasanalternativetoZFNs.ThearchitectureofTALENsissimilartothatofZFNs,withaDNA-bindingdomainfusedtoaFokIdomain(Fig.1).TheDNA-bindingmoduleofTALENsissourcedfromtheDNA-bindingrepeatdomainoftranscriptionactivator-likeeffectors(TALEs),bacterialproteinsoftheplantpathogenXanthomonas(28).Unlikezincfingers,eachTALErepeathasa1-to-1-bpcorrespondence.Mostofthe34aminoacidsineachrepeatarehighlyconserved,exceptfortworepeat-variablediresidues,whichdeterminetheDNAbasespecificity(29,30).TALEsnaturallyexistastandemarraysofrepeatedmotifs,whichmeansthattheyhavebeen“pre-optimized”duringevolutionformodularassembly.ThistranslatesintoauniqueadvantagewhenassemblingTALENs.IncontrasttoZFNs,modularassemblyofTALENshasaessrateupto100%,whichhastwoimportantimplications.1)AnylaboratorywithabasicmolecularbiologysetupcanproducefunctionalTALENswithindays,and2)automatedhigh-throughputTALENproductionispossible(31–33).Indeed,within2shortyearsafterthefirstreportofgenomeeditinginhPSCsusingTALENs,alibraryofTALENstargeting18,740protein-codinggenesinthehumangenomehasalreadybeenconstructedusingahighthroughputcloningmethod(34).InparisonstoZFNs,TALENsparableefficacyandlowertoxicity(35).Asdemonstratedinarecentstudy,TALENshavegreatlyfacilitatedgenomeeditinginhPSCsforgeneratingdiseasemodels(36). DespitetheenthusiasmforTALENs,thetechnologyisstillinitsinfancyandthusfacesmanyunresolvedissues.TALENtarget-siteselectionisrestrictedbytherequirementofaprecedingTbase(29,37).AlthoughthisshouldnotprohibitessfuldesignofTALENsinmostcases,itmaybeanissuewhenmodifyingaspecificmutationespeciallyforfuturecell-basedgenetherapy.ThereportedsensitivityofTALENsto5-methylcytosinecouldbeamoreseriousdrawbackoftheTALENtechnologybecauseoftheprevalenceofthisDNAmodificationinthegenome(38,39).Recentevidenceshowsthatthisproblemmaybeebyengineering5-methylcytosine- insensitiveTALEDNA-bindingdomains(39).Theextentofoff-targeteffectofTALENsislargelyunknown.Recently,therehavebeenseveraleffortstosystematicallymapoff-targetcleavageofZFNsandmeganucleases.Theseincludeinvitroselectionofbindingtargetsbysystemicevolutionofligandsbyexponentialenrichment(SELEX),invitroselectionofcleavagesequence,andtaggingtransientlyappearingDSBsbyNHEJmediatedintegrationofadeno-associatedvirusvectorsorintegrase-defectivelentiviralvectors(21,26,40).ThesemethodsarealsoapplicabletoTALENs.Itisworthnotingthatunbiasedapproachesofmappingoff-targetcleavagesites,suchasthosereportedbyGabrieletal.(21)andPeteketal.(40),showthatinsilicoscreeningofpotentialcleavagetargetsbasedonsequencehomologydoesnotpredictactualcleavageinvivo.Anothermethodofsurveyingoff-targeteffectsofTALENsisgenomesequencing.Dingetal.(36)paredTALEN-modifiedexomeswiththeparentalexomeandfoundlittleevidenceofindels,ahallmarkofTALENcleavagesites.However,theynotedapproximatelysevensingle-nucleotidevariantsperclone(36).Astheauthorssuggested,thesesingle-nucleotidevariantsmayreflectheterogeneityintheparentalpopulation,whichmanifestsitselfduringtheextendedculturethatisrequiredforgenomeediting,astheydonotcoincidewithpredictedTALENoff-targetsites.Usingasinglecell-derivedparentallinemayhelpclarifythisissue.Togainpletepictureofoff-targeteffectsofTALENsinnoncodingregionsofthegenome,highcoveragewhole-genomesequencingisnecessary(36). ClusteredRegularlyInterspacedShortPalindromicRepeat(CRISPR)/CAS9RNA-guidedNucleases—ItisclearfromthedevelopmentofZFNsandTALENsthatspecificsequencerecognitionisthekeyelementinthedesignofsyntheticnucleases.OtherthanDNA-bindingpolypeptides,naturehasevolvedothermeanstointeractwithspecificDNAsequences.BacteriaandarchaeapossessauniqueadaptiveimmunesystembasedonanRNA-guidedDNAendonucleasetodestroyforeignDNA(41).DNAfragmentsofpastinvadersareintegratedasspacersintheCRISPRgenomicloci.TheCRISPRlociaretranscribedtoproduceCRISPRRNAs(crRNAs),whichcontainauniqueseedplementarytotargetDNA(calledprotospacer)andarepeatregionthathybridizestoasmallRNAcalledtransactivatedcrRNA(tracrRNA).ThecrRNAandtracrRNAhybridguidesCAS(CRISPR-associated)proteinstocleavetargetDNA(42).Sincethebeginningof2013,therehasbeenasurgeofreportsofessfuladaptationoftheCRISPR/CAS9systemforhumangenomeediting(43–46).TargetedcleavagebyCRISPR/CAS9inhumancellsrequirestheintroductionofaCAS9expressionvectorandtheguideRNAs(crRNAandtracrRNA).ThesystemhasbeenfurthersimplifiedbyfusingthetwoRNAsintoachimericRNA(44).TargetingCAS9toadesiredgenomicsiteonlyentailscloningthe20bpoftargetsequenceintothespacerregionofthecrRNAlocus(Fig.1).SeveralversionsoftheCRISPR/CAS9systemareavailablefromplasmid-sharingservices.Thispotentiallymakestheexclusivityofgenomeeditingtechnologyathingofthepast.TheCRISPR/CAS9systemisalsoamenabletohigh-throughputconstructionofalibraryoftargetingvectors.BecauseofthesmallsizeoftheguideRNA,itisalsopossibletodelivermultipleguideRNAsatthesametimetoachievemultiplextargeting(45,47).Arecent 4596JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER8•FEBRUARY21,2014 Downloadedfrom/atInstBiophys,CASonJune29,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs studyparedtheefficiencyofCRISPR/CAS9withthatofTALENsintargetingthesamegenomicsitesinhPSCs(48).CAS9outperformedTALENsingenedisruption,geneknockin,andbi-allelictargetingacrossallloci.ItwasspeculatedthatthismightbeduetoahigherexpressionlevelandlowercytotoxicityoftheCAS9protein(48). Despiteitsversatility,theCRISPR/CAS9systemhasseverallimitations.First,thetargetablesitesofCAS9areconstrainedbytherequirementofaGN20GGsequencemotif,whichmaycauseaproblemwhentargetingcertainloci.Second,uptosixmismatchesbetweencrRNAandtargetDNAaretoleratedbyCAS9,whichmayresultinoff-targetcleavage(41).Indeed,arecentstudyshowedthatCRISPR/CAS9nucleasesinducemutationsatoff-targetsiteswithuptofivemismatches(49).Moreimportantly,frequenciesofoff-targetmutationsareequaltoorhigherthanthoseofon-targetmutations(49).CAS9mutantswithamorestringentrequirementofcrRNA-targetplementationmaybeengineeredbydirectedevolution.CAS9hasbeenconvertedintoanickase,whichreducesmutagenesisatoff-targetsites(45).Inaddition,asystematicexaminationoftheoff-targeteffectsofCRISPR/CAS9oughttobeperformedusingthetechnologiesdiscussedabovewithregardtoZFNsandTALENs. GenomeEditingwithoutNucleases Thereareothertoolsthatenableefficientgenomeeditinginhumancellswithouttheaidofsyntheticnucleases.Comparedwithsyntheticnuclease-basedmethods,theclassicHRmethodislesslikelytohaveoff-targeteffects.However,thelowefficiencyofHRinhumancellsisamajorroadblock
(5).Severalapproacheshavebeendesignedtoethisissue,includingtheuseofbacterialartificialchromosomes,adenoassociatedviruses,andhelper-dependentadenoviralvectors(HDAdVs)(50–56).Amongthese,HDAdVshaveenjoyedthemostessingenomeeditingofhPSCs.HDAdVsaresocalledlast-generationadenovirusesdevelopedforgenetherapy.Theyarenon-integrativeviralvectorsengineeredtobelowimmunogenicandabletotransduceawiderangeofcelltypeswithhighefficiency.SinceallviralgenesaredeletedfromHDAdVs,theyhavealargecloningcapacityof36kb(57).ThesefeaturesmakeHDAdVsidealvectorsfordeliveringHRdonorconstructswithextendedhomologyarms(Fig.1).HDAdVshavebeenessfullyappliedtogenomeeditinginhPSCs,includingiccorrectionofHutchinson-Gilfordprogeriasyndrome,sicklecelldisease,andParkinsondiseaseinhiPSCs(50–53).ThepercentageofclonesharboringthecorrecttargetingeventviaHRafterdrugselectionissignificantlyhigherthantheclassicalHRmethod(rangeof17–100%).Unlikesyntheticnucleases,HDAdVsofferaprospectofinvivodelivery.AdditionalbenefitsofHDAdVsinclude1)norestrictionontargetsiteselection,2)simultaneousintroductionofmultiplemodificationstoalargespanofDNAregion,3)efficienttransductionintoawiderangeofcelltypes,and4)noriskofoff-targetcleavage.OnestudyhaslookedatthegenomicandepigenomicstatusofHDAdV-modifiedhiPSCsandfoundittobehighlysimilartothatoftheparentallines(52).However,HDAdVscarryararechanceofintegratingintothegenome,althoughsuchaneventwasnotdetectedbyusingavarietyoftechniquesinour research(50).TheconstructionofHDAdVsisrathertechnicallychallengingandlabor-intensive,whichmaypresentabarriertoadoptingthistechnology.AnotherdrawbackisthatHDAdV-mediatedgenomeeditingrequiresdrugselection,whichislengthyandnecessitatesanadditionalsteptoremovethedrug-selectablemarker,andagenomicscar(e.g.aloxPorFRTsite)isleftbehind.Itispossibletodeliversyntheticnucleases(e.g.TALENs)andthedonorconstructinan“all-inone”HDAdV,thereforeavoidingdrugselectionandtheissuesassociatedwithit. ConclusionsandPerspectives Therapidprogressionofgenomeeditingtechnologyisaboontobothbasicscienceandcellandgenetherapy.Ideally,cellsthathaveundergonegenomeeditingshouldcontainonlytheintendedchangeinanotherwiseisogenicbackground,thusprovidingthemoststringenttestofgenefunction.However,thismaynotbethecaseduetooff-targeteffectsofZFNs,TALENs,andCRISPR/CAS9.Onewaytominimizetheseexperimentalconfoundersistoimprovethedesignofthegenomeeditingtools.DNAnickase,obligateheterodimericFokI,andmutantvariantswithenhancerspecificityallincreasethefidelityofsyntheticnucleases.Becauseitisstilldifficulttodeterminethefullextentofoff-targeteffectsofsyntheticnucleases,prudentexperimentaldesignsshouldbethesecondlineofdefense.Itmaynotbeenoughtojustanalyzemultipleclonesresultingfromonenucleasedesignbecauseoff-targetcleavagecouldmonamongclones.Ifclonesgeneratedbyanindependentnucleasethatistargetedtoadifferentregionofthesamelocushavethesamephenotypes,thenthegenotypephenotyperelationshipcanbeestablishedwithconfidence. Otherthanelucidatinggenefunctionandmodelinghumandiseases,genomeeditingtechnologiescanfacilitateavarietyofnovelstudies,suchasimprovingdirecteddifferentiationofhiPSCsbygeneratinglineage-specificreporterlines,engineeringdendriticcell-directedcancerinesandTcellimmunotherapies(58,59),andgeneratinghumancelllineswithenhancedproductionofbiomoleculesforbiotechnologyandpharmaceuticalindustries. Theuseofgenomeeditingintheclinicrequiresextrascrutiny.Althoughonlyafewexonicmutationsareinducedduringgenecorrection,andmicetransplantedwiththesemodifiedcellsdonothavetumors,thelong-termsafetyissueisstillunclear(27,60).Westilldonotunderstandhowtocontrolmutationumulationduringcultureandtheimplicationsofthesemutationsinvivo.Sincetheseunintendedmutationsarepermanentchangesthatmayhavelong-termdelayedadverseeffects,suchasthoseobservedintheX-linkedbinedimmunodeficiencytrial(25,61),long-termevaluationofthesafetyofcellsthathaveundergonegenomeeditinginprimatesishighlymended.Becausegenomeeditingtechnologyisanemergingfield,itwouldbeadvisablethattheproperauthorities,includingtheFoodandDrugAdministration,issuespecificandsensibleguidelinesforthedesignofpreclinicalstudies.Abetterunderstandingoftheriskofgenomeeditingtechnologiesmayallowtheiruseinnon-life-threateningconditions,thereforepotentiallybenefitingmorepeople.Itmaybeunrealisticandunreasonabletorequireazerotoleranceofmutations FEBRUARY21,2014•VOLUME289•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY4597 Downloadedfrom/atInstBiophys,CASonJune29,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs ordemandafullinvestigationintoeveryicvariance.Perhapsthekeyistostrikeabalancebetweenrisksandbenefitstothepatients.Today,itisclearthatweareexperiencingthedawnofanewerainbiomedicalresearchusheredinbygenomeeditingtechnologies. Acknowledgments—WethankHsin-KaiLiaoandYingGuforhelpfuldiscussions;IlirDubova,MaySchwarz,andPeterSchwarzforadministrativesupport;andJamesA.Cooperforhelpwithillustration. REFERENCES
1.Melton,
D.W.(1994)Genetargetinginthemouse.BioEssays16,633–6382.,
C.,andCohen-Tannoudji,
M.(2001)Genomeengineeringvia homologousbinationinmouseembryonicstem(ES)cells:anamazinglyversatiletoolforthestudyofmammalianbiology.An.Acad.Bras.Cienc.73,365–3833.Sancho-Martinez,
I.,Li,
M.,andIzpisuaBelmonte,
J.C.(2011)DiseasecorrectiontheiPSCway:advancesiniPSC-basedtherapy.Clin.Pharmacol.Ther.89,746–7494.Ruby,
K.M.,andZheng,
B.(2009)GenetargetinginaHUESlineofhumanembryonicstemcellsviaelectroporation.StemCells27,1496–15065.Zwaka,
T.P.,andThomson,
J.A.(2003)Homologousbinationinhumanembryonicstemcells.Nat.Biotechnol.21,319–3216.Bañuelos,
C.A.,Banáth,
J.P.,MacPhail,
S.H.,Zhao,
J.,Eaves,
C.A.,O’Connor,
M.D.,Lansdorp,
P.M.,andOlive,
P.L.(2008)Mousebutnothumanembryonicstemcellsaredeficientinrejoiningofionizingradiation-inducedDNAdouble-strandbreaks.DNARepair7,1471–14837.Meek,
K.,Dang,
V.,andLees-Miller,
S.P.(2008)DNA-PK:themeanstojustifytheends?
Adv.Immunol.99,33–588.Cherry,
A.B.,andDaley,
G.Q.(2013)Reprogrammedcellsfordiseasemodelingandregenerativemedicine.Annu.Rev.Med.64,277–2909.Soldner,
F.,andJaenisch,
R.(2012)Medicine.iPSCdiseasemodeling.Science338,1155–115610.Chapman,
J.R.,Taylor,
M.R.,andBoulton,
S.J.(2012)Playingtheendgame:DNAdouble-strandbreakrepairpathwaychoice.Mol.Cell47,497–51011.Carroll,
D.(2011)Genomeengineeringwithzinc-fingernucleases.ics188,773–78212.Bhakta,
M.S.,Henry,
I.M.,Ousterout,
D.G.,Das,
K.T.,Lockwood,
S.H.,Meckler,
J.F.,Wallen,
M.C.,Zykovich,
A.,Yu,
Y.,Leo,
H.,Xu,
L.,Gersbach,
C.A.,andSegal,
D.J.(2013)Highlyactivezinc-fingernucleasesbyextendedmodularassembly.GenomeRes.23,530–53813.Urnov,
F.D.,Rebar,
E.J.,Holmes,
M.C.,Zhang,
H.S.,andGregory,
P.D.(2010)Genomeeditingwithengineeredzincfingernucleases.Nat.Rev..11,636–64614.Haruta,
M.,Tomita,
Y.,Yuno,
A.,Matsumura,
K.,Ikeda,
T.,Takamatsu,
K.,Haga,
E.,Koba,
C.,Nishimura,
Y.,andSenju,
S.(2013)TAP-deficienthumaniPScell-derivedmyeloidcelllinesasunlimitedcellsourcefordendriticcell-likeantigen-presentingcells.GeneTher.20,504–51315.Chang,
C.J.,andBouhassira,
E.E.(2012)Zinc-fingernuclease-mediatedcorrectionof␣-thalassemiainiPScells.Blood120,3906–391416.Wang,
Y.,Zhang,
W.Y.,Hu,
S.,Lan,
F.,Lee,
A.S.,Huber,
B.,Lisowski,
L.,Liang,
P.,Huang,
M.,deAlmeida,
P.E.,Won,
J.H.,Sun,
N.,Robbins,
R.C.,Kay,
M.A.,Urnov,
F.D.,andWu,
J.C.(2012)Genomeeditingofhumanembryonicstemcellsandinducedpluripotentstemcellswithzincfingernucleasesforcellularimaging.Circ.Res.111,1494–150317.Soldner,
F.,Laganière,
J.,Cheng,
A.W.,Hockemeyer,
D.,Gao,
Q.,Alagappan,
R.,Khurana,
V.,Golbe,
L.I.,Myers,
R.H.,Lindquist,
S.,Zhang,
L.,Guschin,
D.,Fong,
L.K.,Vu,
B.J.,Meng,
X.,Urnov,
F.D.,Rebar,
E.J.,Gregory,
P.D.,Zhang,
H.S.,andJaenisch,
R.(2011)GenerationofisogenicpluripotentstemcellsdifferingexclusivelyattwoearlyonsetParkinsonpointmutations.Cell146,318–33118.Collin,
J.,andLako,
M.(2011)Concisereview:puttingafingeronstemcellbiology:zincfingernuclease-driventargetediceditinginhumanpluripotentstemcells.StemCells29,1021–1033 19.Perez-Pinera,
P.,Ousterout,
D.G.,andGersbach,
C.A.(2012)Advancesintargetedgenomeediting.Curr.Opin.Chem.Biol.16,268–277 20.Ramirez,
C.L.,Foley,
J.E.,Wright,
D.A.,Müller-Lerch,
F.,Rahman,
S.H.,Cornu,
T.I.,Winfrey,
R.J.,Sander,
J.D.,Fu,
F.,Townsend,
J.A.,Cathomen,
T.,Voytas,
D.F.,andJoung,
J.K.(2008)Unexpectedfailureratesformodularassemblyofengineeredzincfingers.Nat.Methods5,374–375 21.Gabriel,
R.,Lombardo,
A.,Arens,
A.,Miller,
J.C.,Genovese,
P.,Kaeppel,
C.,Nowrouzi,
A.,Bartholomae,
C.C.,Wang,
J.,Friedman,
G.,Holmes,
M.C.,Gregory,
P.D.,Glimm,
H.,Schmidt,
M.,Naldini,
L.,andvonKalle,
C.(2011)Anunbiasedgenome-wideanalysisofzinc-fingernucleasespecificity.Nat.Biotechnol.29,816–823 22.Doyon,
Y.,Vo,
T.D.,Mendel,
M.C.,Greenberg,
S.G.,Wang,
J.,Xia,
D.F.,Miller,
J.C.,Urnov,
F.D.,Gregory,
P.D.,andHolmes,
M.C.(2011)Enhancingzinc-finger-nucleaseactivitywithimprovedobligateheterodimericarchitectures.Nat.Methods8,74–79 23.Ramirez,
C.L.,Certo,
M.T.,Mussolino,
C.,Goodwin,
M.J.,Cradick,
T.J.,McCaffrey,
A.P.,Cathomen,
T.,Scharenberg,
A.M.,andJoung,
J.K.(2012)Engineeredzincfingernickasesinducehomology-directedrepairwithreducedmutageniceffects.NucleicAcidsRes.40,5560–5568 24.Wang,
J.,Friedman,
G.,Doyon,
Y.,Wang,
N.S.,Li,
C.J.,Miller,
J.C.,Hua,
K.L.,Yan,
J.J.,Babiarz,
J.E.,Gregory,
P.D.,andHolmes,
M.C.(2012)TargetedgeneadditiontoapredeterminedsiteinthehumangenomeusingaZFN-basednickingenzyme.GenomeRes.22,1316–1326 25.Woods,
N.B.,Bottero,
V.,Schmidt,
M.,vonKalle,
C.,andVerma,
I.M.(2006)Genetherapy:therapeuticgenecausinglymphoma.Nature440,1123 26.Pattanayak,
V.,Ramirez,
C.L.,Joung,
J.K.,andLiu,
D.R.(2011)Revealingoff-targetcleavagespecificitiesofzinc-fingernucleasesbyinvitroselection.Nat.Methods8,765–770 27.Yusa,
K.,Rashid,
S.T.,Strick-Marchand,
H.,Varela,
I.,Liu,
P.Q.,Paschon,
D.E.,Miranda,
E.,Ordóñez,
A.,Hannan,
N.R.,Rouhani,
F.J.,Darche,
S.,Alexander,
G.,Marciniak,
S.J.,Fusaki,
N.,Hasegawa,
M.,Holmes,
M.C.,DiSanto,
J.P.,Lomas,
D.A.,Bradley,
A.,andVallier,
L.(2011)Targetedgenecorrectionof␣1-antitrypsindeficiencyininducedpluripotentstemcells.Nature478,391–394 28.Joung,
J.K.,andSander,
J.D.(2013)TALENs:awidelyapplicabletechnologyfortargetedgenomeediting.Nat.Rev.Mol.CellBiol.14,49–55 29.Boch,
J.,Scholze,
H.,Schornack,
S.,Landgraf,
A.,Hahn,
S.,Kay,
S.,Lahaye,
T.,Nickstadt,
A.,andBonas,
U.(2009)BreakingthecodeofDNAbindingspecificityofTAL-typeIIIeffectors.Science326,1509–1512 30.Mak,
A.N.,Bradley,
P.,Cernadas,
R.A.,Bogdanove,
A.J.,andStoddard,
B.L.(2012)ThecrystalstructureofTALeffectorPthXo1boundtoitsDNAtarget.Science335,716–719 31.Reyon,
D.,Tsai,
S.Q.,Khayter,
C.,Foden,
J.A.,Sander,
J.D.,andJoung,
J.K.(2012)FLASHassemblyofTALENsforhigh-throughputgenomeediting.Nat.Biotechnol.30,460–465 32.Schmid-Burgk,
J.L.,Schmidt,
T.,Kaiser,
V.,Höning,
K.,andHornung,
V.(2013)Aligation-independentcloningtechniqueforhigh-throughputassemblyoftranscriptionactivator-likeeffectorgenes.Nat.Biotechnol.31,76–81 33.Briggs,
A.W.,Rios,
X.,Chari,
R.,Yang,
L.,Zhang,
F.,Mali,
P.,andChurch,
G.M.(2012)Iterativecappedassembly:rapidandscalablesynthesisofrepeat-moduleDNAsuchasTALeffectorsfromindividualmonomers.NucleicAcidsRes.40,e117 34.Kim,
Y.,Kweon,
J.,Kim,
A.,Chon,
J.K.,Yoo,
J.Y.,Kim,
H.J.,Kim,
S.,Lee,
C.,Jeong,
E.,Chung,
E.,Kim,
D.,Lee,
M.S.,Go,
E.M.,Song,
H.J.,Kim,
H.,Cho,
N.,Bang,
D.,Kim,
S.,andKim,
J.S.(2013)AlibraryofTALeffectornucleasesspanningthehumangenome.Nat.Biotechnol.31,251–258 35.Mussolino,
C.,Morbitzer,
R.,Lütge,
F.,Dannemann,
N.,Lahaye,
T.,andCathomen,
T.(2011)AnovelTALEnucleasescaffoldenableshighgenomeeditingactivitybinationwithlowtoxicity.NucleicAcidsRes.39,9283–9293 36.Ding,
Q.,Lee,
Y.K.,Schaefer,
E.A.,Peters,
D.T.,Veres,
A.,Kim,
K.,Kuperwasser,
N.,Motola,
D.L.,Meissner,
T.B.,Hendriks,
W.T.,Trevisan,
M.,Gupta,
R.M.,Moisan,
A.,Banks,
E.,Friesen,
M.,Schinzel,
R.T.,Xia,
F.,Tang,
A.,Xia,
Y.,Figueroa,
E.,Wann,
A.,Ahfeldt,
T.,Daheron,
L.,Zhang,
F.,Rubin,
L.L.,Peng,
L.F.,Chung,
R.T.,Musunuru,
K.,andCowan,
C.A.(2013)ATALENgenome-editingsystemforgeneratinghumanstemcell- 4598JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER8•FEBRUARY21,2014 Downloadedfrom/atInstBiophys,CASonJune29,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs baseddiseasemodels.CellStemCell12,238–25137.Moscou,
M.J.,andBogdanove,
A.J.(2009)AsimpleciphergovernsDNA recognitionbyTALeffectors.Science326,150138.Bultmann,
S.,Morbitzer,
R.,Schmidt,
C.S.,Thanisch,
K.,Spada,
F.,El- saesser,
J.,Lahaye,
T.,andLeonhardt,
H.(2012)Targetedtranscriptionalactivationofsilentoct4pluripotencygenebiningdesignerTALEsandinhibitionoficmodifiers.NucleicAcidsRes.40,5368–537739.Valton,
J.,Dupuy,
A.,Daboussi,
F.,Thomas,
S.,Maréchal,
A.,Macmaster,
R.,Melliand,
K.,Juillerat,
A.,andDuchateau,
P.(2012)ingtranscriptionactivator-likeeffector(TALE)DNAbindingdomainsensitivitytocytosinemethylation.J.Biol.Chem.287,38427–3843240.Petek,
L.M.,Russell,
D.W.,andMiller,
D.G.(2010)Frequentendonucleasecleavageatoff-targetlocationsinvivo.Mol.Ther.18,983–98641.Jinek,
M.,Chylinski,
K.,Fonfara,
I.,Hauer,
M.,Doudna,
J.A.,andCharpentier,
E.(2012)Aprogrammabledual-RNA-guidedDNAendonucleaseinadaptivebacterialimmunity.Science337,816–82142.Wiedenheft,
B.,Sternberg,
S.H.,andDoudna,
J.A.(2012)RNA-guidedicsilencingsystemsinbacteriaandarchaea.Nature482,331–33843.Jinek,
M.,East,
A.,Cheng,
A.,Lin,
S.,Ma,
E.,andDoudna,
J.(2013)RNAprogrammedgenomeeditinginhumancells.eLife2,e0047144.Cho,
S.W.,Kim,
S.,Kim,
J.M.,andKim,
J.S.(2013)TargetedgenomeengineeringinhumancellswiththeCas9RNA-guidedendonuclease.Nat.Biotechnol.31,230–23245.Cong,
L.,Ran,
F.A.,Cox,
D.,Lin,
S.,Barretto,
R.,Habib,
N.,Hsu,
P.D.,Wu,
X.,Jiang,
W.,Marraffini,
L.A.,andZhang,
F.(2013)MultiplexgenomeengineeringusingCRISPR/Cassystems.Science339,819–82346.Mali,
P.,Yang,
L.,Esvelt,
K.M.,Aach,
J.,Guell,
M.,DiCarlo,
J.E.,Norville,
J.E.,andChurch,
G.M.(2013)RNA-guidedhumangenomeengineeringviaCas9.Science339,823–82647.Wang,
H.,Yang,
H.,Shivalila,
C.S.,Dawlaty,
M.M.,Cheng,
A.W.,Zhang,
F.,andJaenisch,
R.(2013)One-stepgenerationofmicecarryingmutationsinmultiplegenesbyCRISPR/Cas-mediatedgenomeengineering.Cell153,910–91848.Ding,
Q.,Regan,
S.N.,Xia,
Y.,Oostrom,
L.A.,Cowan,
C.A.,andMusunuru,
K.(2013)EnhancedefficiencyofhumanpluripotentstemcellgenomeeditingthroughreplacingTALENswithCRISPRs.CellStemCell12,393–39449.Fu,
Y.,Foden,
J.A.,Khayter,
C.,Maeder,
M.L.,Reyon,
D.,Joung,
J.K.,andSander,
J.D.(2013)High-frequencyoff-targetmutagenesisinducedbyCRISPR-Casnucleasesinhumancells.Nat.Biotechnol.31,822–82650.Li,
M.,Suzuki,
K.,Qu,
J.,Saini,
P.,Dubova,
I.,Yi,
F.,Lee,
J.,Sancho-Martinez,
I.,Liu,
G.H.,andIzpisuaBelmonte,
J.C.(2011)Efficientcorrectionofhemoglobinopathy-causingmutationsbyhomologousbinationinintegration-freepatientiPSCs.CellRes.21,1740–174451.Liu,
G.H.,Qu,
J.,Suzuki,
K.,Nivet,
E.,Li,
M.,Montserrat,
N.,Yi,
F.,Xu,
X.,Ruiz,
S.,Zhang,
W.,Wagner,
U.,Kim,
A.,Ren,
B.,Li,
Y.,Goebl,
A.,Kim,
J.,Soligalla,
R.D.,Dubova,
I.,Thompson,
J.,Yates,
J.,3rd,Esteban,
C.R.,Sancho-Martinez,
I.,andIzpisuaBelmonte,
J.C.(2012)ProgressivedegenerationofhumanneuralstemcellscausedbypathogenicLRRK2.Nature491,603–607 52.Liu,
G.H.,Suzuki,
K.,Qu,
J.,Sancho-Martinez,
I.,Yi,
F.,Li,
M.,Kumar,
S.,Nivet,
E.,Kim,
J.,Soligalla,
R.D.,Dubova,
I.,Goebl,
A.,Plongthongkum,
N.,Fung,
H.L.,Zhang,
K.,Loring,
J.F.,Laurent,
L.C.,andIzpisuaBelmonte,
J.C.(2011)Targetedgenecorrectionoflaminopathy-associatedLMNAmutationsinpatient-specificiPSCs.CellStemCell8,688–694 53.Suzuki,
K.,Mitsui,
K.,Aizawa,
E.,Hasegawa,
K.,Kawase,
E.,Yamagishi,
T.,Shimizu,
Y.,Suemori,
H.,Nakatsuji,
N.,andMitani,
K.(2008)Highlyefficienttransientgeneexpressionandgenetargetinginprimateembryonicstemcellswithhelper-dependentadenoviralvectors.Proc.Natl.Acad.Sci.U.S.A.105,13781–13786 54.Mitsui,
K.,Suzuki,
K.,Aizawa,
E.,Kawase,
E.,Suemori,
H.,Nakatsuji,
N.,andMitani,
K.(2009)Genetargetinginhumanpluripotentstemcellswithadeno-associatedvirusvectors.Biochem.Biophys.Res.Commun.388,711–717 55.Khan,
I.F.,Hirata,
R.K.,Wang,
P.R.,Li,
Y.,Kho,
J.,Nelson,
A.,Huo,
Y.,Zavaljevski,
M.,Ware,
C.,andRussell,
D.W.(2010)EngineeringofhumanpluripotentstemcellsbyAAV-mediatedgenetargeting.Mol.Ther.18,1192–1199 56.Song,
H.,Chung,
S.K.,andXu,
Y.(2010)ModelingdiseaseinhumanESCsusinganefficientBAC-basedhomologousbinationsystem.CellStemCell6,80–89 57.Alba,
R.,Bosch,
A.,andChillon,
M.(2005)Gutlessadenovirus:last-generationadenovirusforgenetherapy.GeneTher.12,S18–S27 58.Qu,
X.,Wang,
P.,Ding,
D.,Li,
L.,Wang,
H.,Ma,
L.,Zhou,
X.,Liu,
S.,Lin,
S.,Wang,
X.,Zhang,
G.,Liu,
S.,Liu,
L.,Wang,
J.,Zhang,
F.,Lu,
D.,andZhu,
H.(2013)Zinc-finger-nucleasesmediatespecificandefficientexcisionofHIV-1proviralDNAfrominfectedandlatentlyinfectedhumanTcells.NucleicAcidsRes.41,7771–7782 59.Provasi,
E.,Genovese,
P.,Lombardo,
A.,Magnani,
Z.,Liu,
P.Q.,Reik,
A.,Chu,
V.,Paschon,
D.E.,Zhang,
L.,Kuball,
J.,Camisa,
B.,Bondanza,
A.,Casorati,
G.,Ponzoni,
M.,Ciceri,
F.,Bordignon,
C.,Greenberg,
P.D.,Holmes,
M.C.,Gregory,
P.D.,Naldini,
L.,andBonini,
C.(2012)EditingTcellspecificitytowardsleukemiabyzincfingernucleasesandlentiviralgenetransfer.Nat.Med.18,807–815 60.Howden,
S.E.,Gore,
A.,Li,
Z.,Fung,
H.L.,Nisler,
B.S.,Nie,
J.,Chen,
G.,McIntosh,
B.E.,Gulbranson,
D.R.,Diol,
N.R.,Taapken,
S.M.,Vereide,
D.T.,Montgomery,
K.D.,Zhang,
K.,Gamm,
D.M.,andThomson,
J.A.(2011)iccorrectionandanalysisofinducedpluripotentstemcellsfromapatientwithgyrateatrophy.Proc.Natl.Acad.Sci.U.S.A.108,6537–6542 61.Hacein-Bey-Abina,
S.,VonKalle,
C.,Schmidt,
M.,McCormack,
M.P.,Wulffraat,
N.,Leboulch,
P.,Lim,
A.,Osborne,
C.S.,Pawliuk,
R.,Morillon,
E.,Sorensen,
R.,Forster,
A.,Fraser,
P.,Cohen,
J.I.,deSaintBasile,
G.,Alexander,
I.,Wintergerst,
U.,Frebourg,
T.,Aurias,
A.,,
D.,Romana,
S.,Radford-Weiss,
I.,Gross,
F.,Valensi,
F.,Delabesse,
E.,Macintyre,
E.,Sigaux,
F.,Soulier,
J.,Leiva,
L.E.,Wissler,
M.,Prinz,
C.,Rabbitts,
T.H.,LeDeist,
F.,Fischer,
A.,andCavazzana-Calvo,
M.(2003)LMO2-associatedclonalTcellproliferationintwopatientsaftergenetherapyforSCID-X1.Science302,415–419 FEBRUARY21,2014•VOLUME289•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY4599
J.Biol.Chem.2014,289:4594-4599.doi:10.1074/jbc.R113.488247originallypublishedonlineDecember20,2013 essthemostupdatedversionofthisarticleatdoi:10.1074/jbc.R113.488247 Findarticles,minireviews,ReflectionsandClassicsonicsontheJBCAffinitySites. Alerts:•Whenthisarticleiscited•Whenacorrectionforthisarticleisposted ClickheretochoosefromallofJBC'se-mailalerts Thisarticlecites61references,22ofwhichcanbeessedfreeat#ref-list-
1 MINIREVIEW THEJOURNALOFBIOLOGICALCHEMISTRYVOL.289,NO.8,pp.4594–4599,February21,2014©2014byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PublishedintheU.S.A. Downloadedfrom/atInstBiophys,CASonJune29,2014 ACutabovetheRest:TargetedGenomeEditingTechnologiesinHumanPluripotentStemCells* Published,JBCPapersinPress,December20,2013,DOI10.1074/jbc.R113.488247 MoLi‡1,
2,KeiichiroSuzuki‡1,
2,NaYoungKim‡,Guang-HuiLiu§3,andJuanCarlosIzpisuaBelmonte‡4Fromthe‡GeneExpressionLaboratory,SalkInstituteforBiologicalStudies,LaJolla,California92037andthe§NationalLaboratoryofBiomacromolecules,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,China Humanpluripotentstemcells(hPSCs)offerunprecedentedopportunitiestostudycellulardifferentiationandmodelhumandiseases.TheabilitytopreciselymodifyanygenomicsequenceholdsthekeytorealizingthefullpotentialofhPSCs.ThankstotherapiddevelopmentofnovelgenomeeditingtechnologiesdrivenbytheenormousinterestinthehPSCfield,genomeeditinginhPSCshasevolvedfrombeingadauntingtaskafewyearsagotoaroutineprocedureinmostlaboratories.Here,weprovideanoverviewofthemainstreamgenomeeditingtools,includingzincfingernucleases,transcriptionactivator-likeeffectornucleases,clusteredregularlyinterspacedshortpalindromicrepeat/CAS9RNA-guidednucleases,andhelper-dependentadenoviralvectors.Wediscussthefeaturesandlimitationsofthesetechnologies,aswellashowthesefactorsinfluencetheutilityofthesetoolsinbasicresearchandtherapies. Thediscoveryofinducedpluripotentstemcells(iPSCs)5sevenyearsagohasreignitedtheenthusiasmforcell-basedtherapy.TheabilityofiPSCstoundergounlimiteddivisionwhilemaintaininggenomicintegrityprovidesawaytoethesenescencebarrierofagedsomaticcells.ThecapacityofiPSCstodifferentiateintocellsofthethreegermlayershasbeenextensivelydocumentedinthefield.Takentogether,itisnothardtoappreciatewhyhumaniPSC(hiPSC)-basedautolo- *ThisworkwassupportedinpartbygrantsfromtheG.HaroldandLeilaY. MathersCharitableFoundation,theEllisonMedicalFoundation,theLeonaM.andHarryB.HelmsleyCharitableTrust,andtheGlennFoundationforMedicalResearch(toJ.C.I.B.).ThisisthesixtharticleintheThematicMinireviewSeries“DevelopmentofHumanTherapeuticsBasedonInducedPluripotentStemCell(iPSC)Technology.”1Bothauthorscontributedequallytothiswork.2SupportedbyfellowshipsfromtheCaliforniaInstituteofRegenerativeMedicine.3Supportedbythe“ThousandYoungTalents”programofChina,theNationalLaboratoryofBiomacromolecules,andtheStrategicPriorityResearchProgramoftheChineseAcademyofSciences.4Towhomcorrespondenceshouldbeaddressed.E-mail:belmonte@salk.edu.5Theabbreviationsusedare:iPSC,inducedpluripotentstemcell;hiPSC,humaniPSC;HR,homologousbination;hESC,humanembryonicstemcell;DSB,DNAdouble-strandbreak;NHEJ,non-homologousendjoining;HDR,homology-directedrepair;ZFN,zincfingernuclease;TALEN,transcriptionactivator-likeeffectornuclease;TALE,transcriptionactivatorlikeeffector;CRISPR,clusteredregularlyinterspacedshortpalindromicrepeat;crRNA,CRISPRRNA;tracrRNA,transactivatedcrRNA;HDAdV,helper-dependentadenoviralvector. goustransplantationisheraldedasthefutureofregenerativemedicine(Fig.1).Oneareathathasdrawngreatinterestiscorrectionoficdiseasesinpatient-specifichiPSCswithaprospectofpersonalizedcelltherapy. Pluripotentstemcellsareespeciallyamenableforgenomeeditingbecausetheycanundergoextensivetissueculturemanipulations,suchasdrugselectionandclonalexpansion,whilestillmaintainingtheirpluripotencyandgenomestability.Genetargetinginmouseembryonicstemcellsbyhomologousbination(HR)hasproventobeastapletechniqueforstudyinggenefunction(1,2).However,thesamestrategydoesnottranslatewellintohumanembryonicstemcells(hESCs)orhiPSCs
(3).AlthoughtheclassicalHRmethodhasbeenessfullyusedtogenerateknock-inreporterlinesandtocorrectgenemutationsinhESCs,thereportedtargetingefficienciesareatleastseveralordersofmagnitudelowerthanwhatisachievableinmouseembryonicstemcells(4,5).ThisislikelyduetotheintrinsicdifferencesintheDNArepairprocessbetweenhumansandmice,asmeasurestoimprovesingle-cellsurvivalandDNAtransfectiondidnothaveadramaticeffectongenetargetingefficiency(6,7).However,othermethodsaimedatpromotingHRprovedmorefruitful
(3).Inthepastseveralyears,therehasbeenaspikeofinterestingenomeeditinginhESCsandhiPSCs,possiblyduetothepotentialofthistechnologyinmodelingandcorrectingamyriadoficdiseases(8,9).Thishasfueledarapiddevelopmentinnoveltechnologiesfortargetedmodificationofthehumangenome.Here,weaimtoprovideatimelyupdateonthecurrentgenomeeditingtechnologiesanddiscussthefactorsthatinfluencethechoiceofanappropriatetechnology. GenomeEditingwithSyntheticNucleases IntroductionofaDNAdouble-strandbreak(DSB)triggersDNArepairresponsesviatwomajorpathways(10).TheDSBsarerepairedprimarilybythenon-homologousendjoining(NHEJ)pathway,whicheitherrestorestheoriginalsequenceorcreatessmallinsertionsordeletions(indels).Alternatively,theDSBsmayberepairedthroughHRbasedonahomologousrepairtemplate(aprocesstermedhomology-directedrepair(HDR)).HDRcanbeco-optedtointroducedesiredsequencechangeswhenanexogenoustemplateisintroduced.Therehavebeenseveralapproachesofengineeringsyntheticnucleasestoachievetargetedcleavageofaspecificsiteinthehumangenome.Thesenucleasessharetwoimportantproperties:1)amechanismofrecognizingasufficientlylongtargetsequencethatursonlyonceinthegenomeand2)acatalyticactivitythatisactivatedbysequence-specificbinding. ZincFingerNucleases—Zincfingernucleases(ZFNs)aremodularproteinsconsistingofaseriesoftheCys2-His2zincfingerDNA-bindingmotifsandtheDNAcleavagedomainofthetypeIISrestrictionenzymeFokI(11).Eachzincfingermotifrecognizes3–4bpofDNA,andthemodularnatureofthezincfingermotifallowsspecificbindingtopositesequencebylinkingseveralmotifsintandem.TheactivityoftheFokInucleaserequiresdimerization.Therefore,twoZFNsare 4594JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER8•FEBRUARY21,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs Downloadedfrom/atInstBiophys,CASonJune29,2014 FIGURE1.ConceptualschematicshowingtheapplicationofgenomeeditingandiPSCtechnologiesinregenerativemedicine. designedtobindonoppositesidesofthetargetsitewiththerespectiveFokIdomainsorientedtowardeachother(Fig.1).ThisdesignfurtherenhancesthespecificityofZFNs,asdimerizationofFokIandthuscleavagearedependentonalongertarget.ApairofZFNsthateachcontainsthreezincfingermotifsaresufficienttoensureauniqueintendedtargetofan18-bpsequencebychanceinthehumangenome,althoughfourtosixmotifsaremonlyusedtoincreasespecificity(12).ZFNshavebeenessfullyusedtoeditthegenomeofanisms,includinghumans(11,13). Amongthesyntheticnucleases,ZFNshavetheadvantageofbeingthemoststudied.TherearemanytechnicalresourcestoaidthedesignandassemblyofZFNs.WelldesignedZFNsarehighlyeffectiveinthedisruption,addition,orcorrectionofthetargetgene.Recently,ZFN-mediateddisruptionoftheTAP2geneinhiPSCsmadeitpossibletoproduceanunlimitedamountofantigen-presentingcellsforinationtherapy(14).ZFN-mediatedinsertionoftransgenesintothegenomicsafeharborlocus(AAVS1)ofhiPSCshasbeenusedtoengineercellsforinvivoimagingandtocorrecttheglobinimbalancein␣-thalassemia(15,16).InthecaseofHDR,ZFNsgreatlysimplifytheexperimentaldesign,asshortoligonucleotidesmaybeusedastemplates,andantibioticselectionmaybeomitted(17). ForadetailedountofgenomeeditingofhESCsandhiPSCsusingZFNs,wewouldliketoreferthereaderstorecentreviews(18,19). DespitemanyessfulreportsofZFN-mediatedgenomeeditingofhumancellsinacademicandclinicalresearch,ZFNtechnologyhasseverallimitations.Theassemblyofzincfingermotifsisnotmodularinthestrictestsense.Thebindingaffinityofindividualmotifsiscontext-dependent.Inotherwords,anassembledZFNdoesnotnecessarilyhavehighaffinityforthesequencethatispositeofthe3-bpcognatesequenceofeachzincfingermotif(20).Otherselection-basedmethodshavebeeninventedtoaddressthehighfailurerateofmodularlyassembledZFNs(reviewedinRef.14).Withthesemethods,thenumberoftargetablesitesisreduced.Inanycase,itrequiresaconsiderableamountofexperience,time,andefforttoachieveproficiencyatmakingfunctionalZFNs. Unintendedcleavageatso-calledoff-targetsitesisanotherconcernwithZFNs(21).Off-targetcleavagecouldcausecytotoxicity,introduceunknownmutations,andconfoundtheanalysisoftheeffectsoftheintendedicchanges.FokIvariantsthatformobligateheterodimershavebeenusedtominimizeoff-targetcuttingduetohomodimerizationofZFNs(22).ConvertingFokIintoaDNA-nickingenzymealsohelpsto FEBRUARY21,2014•VOLUME289•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY4595 Downloadedfrom/atInstBiophys,CASonJune29,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs reducetheriskofmutagenesisduetooff-targetcleavagebecausesingle-strandbreaksstimulateHDRontargetwhileminimizingNHEJofftarget(23,24).Evenwiththesetechnicalimprovements,itisstillimportanttomonitoroff-targetcleavageofZFNsintendedfortherapeutics.Bioinformaticstoolsmaybeusedtopredictpotentialoff-targetsites.However,thisapproachdoesnottakeintoconsiderationthedifferencesbetweenthetargetgenomeandthereferencegenome.Furthermore,twostudieshaveshownthattheinvivooff-targetsitesofZFNcleavagecouldnotbefullypredictedbyinvitroorinsilicoanalysis(21,26).Atthecellularlevel,although␥H2AXandp53BP1fociareusedtomonitorDSBsitesaftertheintroductionofZFNs,theycannotdistinguishZFN-mediatedDSBsandspontaneousDSBs.Amoredirectapproachwillbesequencingthegenome,whichisingmorepracticalasnextgenerationsequencingesmoreaffordable.Encouragingly,Yusaetal.(27)foundnoevidenceofoff-targetcleavage-inducedmutationinaZFN-modifiedhiPSClinebyexomesequencing. TranscriptionActivator-likeEffectorNucleases—Recently,anothersyntheticnucleasetermedtranscriptionactivator-likeeffectornuclease(TALEN)hasemergedasanalternativetoZFNs.ThearchitectureofTALENsissimilartothatofZFNs,withaDNA-bindingdomainfusedtoaFokIdomain(Fig.1).TheDNA-bindingmoduleofTALENsissourcedfromtheDNA-bindingrepeatdomainoftranscriptionactivator-likeeffectors(TALEs),bacterialproteinsoftheplantpathogenXanthomonas(28).Unlikezincfingers,eachTALErepeathasa1-to-1-bpcorrespondence.Mostofthe34aminoacidsineachrepeatarehighlyconserved,exceptfortworepeat-variablediresidues,whichdeterminetheDNAbasespecificity(29,30).TALEsnaturallyexistastandemarraysofrepeatedmotifs,whichmeansthattheyhavebeen“pre-optimized”duringevolutionformodularassembly.ThistranslatesintoauniqueadvantagewhenassemblingTALENs.IncontrasttoZFNs,modularassemblyofTALENshasaessrateupto100%,whichhastwoimportantimplications.1)AnylaboratorywithabasicmolecularbiologysetupcanproducefunctionalTALENswithindays,and2)automatedhigh-throughputTALENproductionispossible(31–33).Indeed,within2shortyearsafterthefirstreportofgenomeeditinginhPSCsusingTALENs,alibraryofTALENstargeting18,740protein-codinggenesinthehumangenomehasalreadybeenconstructedusingahighthroughputcloningmethod(34).InparisonstoZFNs,TALENsparableefficacyandlowertoxicity(35).Asdemonstratedinarecentstudy,TALENshavegreatlyfacilitatedgenomeeditinginhPSCsforgeneratingdiseasemodels(36). DespitetheenthusiasmforTALENs,thetechnologyisstillinitsinfancyandthusfacesmanyunresolvedissues.TALENtarget-siteselectionisrestrictedbytherequirementofaprecedingTbase(29,37).AlthoughthisshouldnotprohibitessfuldesignofTALENsinmostcases,itmaybeanissuewhenmodifyingaspecificmutationespeciallyforfuturecell-basedgenetherapy.ThereportedsensitivityofTALENsto5-methylcytosinecouldbeamoreseriousdrawbackoftheTALENtechnologybecauseoftheprevalenceofthisDNAmodificationinthegenome(38,39).Recentevidenceshowsthatthisproblemmaybeebyengineering5-methylcytosine- insensitiveTALEDNA-bindingdomains(39).Theextentofoff-targeteffectofTALENsislargelyunknown.Recently,therehavebeenseveraleffortstosystematicallymapoff-targetcleavageofZFNsandmeganucleases.Theseincludeinvitroselectionofbindingtargetsbysystemicevolutionofligandsbyexponentialenrichment(SELEX),invitroselectionofcleavagesequence,andtaggingtransientlyappearingDSBsbyNHEJmediatedintegrationofadeno-associatedvirusvectorsorintegrase-defectivelentiviralvectors(21,26,40).ThesemethodsarealsoapplicabletoTALENs.Itisworthnotingthatunbiasedapproachesofmappingoff-targetcleavagesites,suchasthosereportedbyGabrieletal.(21)andPeteketal.(40),showthatinsilicoscreeningofpotentialcleavagetargetsbasedonsequencehomologydoesnotpredictactualcleavageinvivo.Anothermethodofsurveyingoff-targeteffectsofTALENsisgenomesequencing.Dingetal.(36)paredTALEN-modifiedexomeswiththeparentalexomeandfoundlittleevidenceofindels,ahallmarkofTALENcleavagesites.However,theynotedapproximatelysevensingle-nucleotidevariantsperclone(36).Astheauthorssuggested,thesesingle-nucleotidevariantsmayreflectheterogeneityintheparentalpopulation,whichmanifestsitselfduringtheextendedculturethatisrequiredforgenomeediting,astheydonotcoincidewithpredictedTALENoff-targetsites.Usingasinglecell-derivedparentallinemayhelpclarifythisissue.Togainpletepictureofoff-targeteffectsofTALENsinnoncodingregionsofthegenome,highcoveragewhole-genomesequencingisnecessary(36). ClusteredRegularlyInterspacedShortPalindromicRepeat(CRISPR)/CAS9RNA-guidedNucleases—ItisclearfromthedevelopmentofZFNsandTALENsthatspecificsequencerecognitionisthekeyelementinthedesignofsyntheticnucleases.OtherthanDNA-bindingpolypeptides,naturehasevolvedothermeanstointeractwithspecificDNAsequences.BacteriaandarchaeapossessauniqueadaptiveimmunesystembasedonanRNA-guidedDNAendonucleasetodestroyforeignDNA(41).DNAfragmentsofpastinvadersareintegratedasspacersintheCRISPRgenomicloci.TheCRISPRlociaretranscribedtoproduceCRISPRRNAs(crRNAs),whichcontainauniqueseedplementarytotargetDNA(calledprotospacer)andarepeatregionthathybridizestoasmallRNAcalledtransactivatedcrRNA(tracrRNA).ThecrRNAandtracrRNAhybridguidesCAS(CRISPR-associated)proteinstocleavetargetDNA(42).Sincethebeginningof2013,therehasbeenasurgeofreportsofessfuladaptationoftheCRISPR/CAS9systemforhumangenomeediting(43–46).TargetedcleavagebyCRISPR/CAS9inhumancellsrequirestheintroductionofaCAS9expressionvectorandtheguideRNAs(crRNAandtracrRNA).ThesystemhasbeenfurthersimplifiedbyfusingthetwoRNAsintoachimericRNA(44).TargetingCAS9toadesiredgenomicsiteonlyentailscloningthe20bpoftargetsequenceintothespacerregionofthecrRNAlocus(Fig.1).SeveralversionsoftheCRISPR/CAS9systemareavailablefromplasmid-sharingservices.Thispotentiallymakestheexclusivityofgenomeeditingtechnologyathingofthepast.TheCRISPR/CAS9systemisalsoamenabletohigh-throughputconstructionofalibraryoftargetingvectors.BecauseofthesmallsizeoftheguideRNA,itisalsopossibletodelivermultipleguideRNAsatthesametimetoachievemultiplextargeting(45,47).Arecent 4596JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER8•FEBRUARY21,2014 Downloadedfrom/atInstBiophys,CASonJune29,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs studyparedtheefficiencyofCRISPR/CAS9withthatofTALENsintargetingthesamegenomicsitesinhPSCs(48).CAS9outperformedTALENsingenedisruption,geneknockin,andbi-allelictargetingacrossallloci.ItwasspeculatedthatthismightbeduetoahigherexpressionlevelandlowercytotoxicityoftheCAS9protein(48). Despiteitsversatility,theCRISPR/CAS9systemhasseverallimitations.First,thetargetablesitesofCAS9areconstrainedbytherequirementofaGN20GGsequencemotif,whichmaycauseaproblemwhentargetingcertainloci.Second,uptosixmismatchesbetweencrRNAandtargetDNAaretoleratedbyCAS9,whichmayresultinoff-targetcleavage(41).Indeed,arecentstudyshowedthatCRISPR/CAS9nucleasesinducemutationsatoff-targetsiteswithuptofivemismatches(49).Moreimportantly,frequenciesofoff-targetmutationsareequaltoorhigherthanthoseofon-targetmutations(49).CAS9mutantswithamorestringentrequirementofcrRNA-targetplementationmaybeengineeredbydirectedevolution.CAS9hasbeenconvertedintoanickase,whichreducesmutagenesisatoff-targetsites(45).Inaddition,asystematicexaminationoftheoff-targeteffectsofCRISPR/CAS9oughttobeperformedusingthetechnologiesdiscussedabovewithregardtoZFNsandTALENs. GenomeEditingwithoutNucleases Thereareothertoolsthatenableefficientgenomeeditinginhumancellswithouttheaidofsyntheticnucleases.Comparedwithsyntheticnuclease-basedmethods,theclassicHRmethodislesslikelytohaveoff-targeteffects.However,thelowefficiencyofHRinhumancellsisamajorroadblock
(5).Severalapproacheshavebeendesignedtoethisissue,includingtheuseofbacterialartificialchromosomes,adenoassociatedviruses,andhelper-dependentadenoviralvectors(HDAdVs)(50–56).Amongthese,HDAdVshaveenjoyedthemostessingenomeeditingofhPSCs.HDAdVsaresocalledlast-generationadenovirusesdevelopedforgenetherapy.Theyarenon-integrativeviralvectorsengineeredtobelowimmunogenicandabletotransduceawiderangeofcelltypeswithhighefficiency.SinceallviralgenesaredeletedfromHDAdVs,theyhavealargecloningcapacityof36kb(57).ThesefeaturesmakeHDAdVsidealvectorsfordeliveringHRdonorconstructswithextendedhomologyarms(Fig.1).HDAdVshavebeenessfullyappliedtogenomeeditinginhPSCs,includingiccorrectionofHutchinson-Gilfordprogeriasyndrome,sicklecelldisease,andParkinsondiseaseinhiPSCs(50–53).ThepercentageofclonesharboringthecorrecttargetingeventviaHRafterdrugselectionissignificantlyhigherthantheclassicalHRmethod(rangeof17–100%).Unlikesyntheticnucleases,HDAdVsofferaprospectofinvivodelivery.AdditionalbenefitsofHDAdVsinclude1)norestrictionontargetsiteselection,2)simultaneousintroductionofmultiplemodificationstoalargespanofDNAregion,3)efficienttransductionintoawiderangeofcelltypes,and4)noriskofoff-targetcleavage.OnestudyhaslookedatthegenomicandepigenomicstatusofHDAdV-modifiedhiPSCsandfoundittobehighlysimilartothatoftheparentallines(52).However,HDAdVscarryararechanceofintegratingintothegenome,althoughsuchaneventwasnotdetectedbyusingavarietyoftechniquesinour research(50).TheconstructionofHDAdVsisrathertechnicallychallengingandlabor-intensive,whichmaypresentabarriertoadoptingthistechnology.AnotherdrawbackisthatHDAdV-mediatedgenomeeditingrequiresdrugselection,whichislengthyandnecessitatesanadditionalsteptoremovethedrug-selectablemarker,andagenomicscar(e.g.aloxPorFRTsite)isleftbehind.Itispossibletodeliversyntheticnucleases(e.g.TALENs)andthedonorconstructinan“all-inone”HDAdV,thereforeavoidingdrugselectionandtheissuesassociatedwithit. ConclusionsandPerspectives Therapidprogressionofgenomeeditingtechnologyisaboontobothbasicscienceandcellandgenetherapy.Ideally,cellsthathaveundergonegenomeeditingshouldcontainonlytheintendedchangeinanotherwiseisogenicbackground,thusprovidingthemoststringenttestofgenefunction.However,thismaynotbethecaseduetooff-targeteffectsofZFNs,TALENs,andCRISPR/CAS9.Onewaytominimizetheseexperimentalconfoundersistoimprovethedesignofthegenomeeditingtools.DNAnickase,obligateheterodimericFokI,andmutantvariantswithenhancerspecificityallincreasethefidelityofsyntheticnucleases.Becauseitisstilldifficulttodeterminethefullextentofoff-targeteffectsofsyntheticnucleases,prudentexperimentaldesignsshouldbethesecondlineofdefense.Itmaynotbeenoughtojustanalyzemultipleclonesresultingfromonenucleasedesignbecauseoff-targetcleavagecouldmonamongclones.Ifclonesgeneratedbyanindependentnucleasethatistargetedtoadifferentregionofthesamelocushavethesamephenotypes,thenthegenotypephenotyperelationshipcanbeestablishedwithconfidence. Otherthanelucidatinggenefunctionandmodelinghumandiseases,genomeeditingtechnologiescanfacilitateavarietyofnovelstudies,suchasimprovingdirecteddifferentiationofhiPSCsbygeneratinglineage-specificreporterlines,engineeringdendriticcell-directedcancerinesandTcellimmunotherapies(58,59),andgeneratinghumancelllineswithenhancedproductionofbiomoleculesforbiotechnologyandpharmaceuticalindustries. Theuseofgenomeeditingintheclinicrequiresextrascrutiny.Althoughonlyafewexonicmutationsareinducedduringgenecorrection,andmicetransplantedwiththesemodifiedcellsdonothavetumors,thelong-termsafetyissueisstillunclear(27,60).Westilldonotunderstandhowtocontrolmutationumulationduringcultureandtheimplicationsofthesemutationsinvivo.Sincetheseunintendedmutationsarepermanentchangesthatmayhavelong-termdelayedadverseeffects,suchasthoseobservedintheX-linkedbinedimmunodeficiencytrial(25,61),long-termevaluationofthesafetyofcellsthathaveundergonegenomeeditinginprimatesishighlymended.Becausegenomeeditingtechnologyisanemergingfield,itwouldbeadvisablethattheproperauthorities,includingtheFoodandDrugAdministration,issuespecificandsensibleguidelinesforthedesignofpreclinicalstudies.Abetterunderstandingoftheriskofgenomeeditingtechnologiesmayallowtheiruseinnon-life-threateningconditions,thereforepotentiallybenefitingmorepeople.Itmaybeunrealisticandunreasonabletorequireazerotoleranceofmutations FEBRUARY21,2014•VOLUME289•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY4597 Downloadedfrom/atInstBiophys,CASonJune29,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs ordemandafullinvestigationintoeveryicvariance.Perhapsthekeyistostrikeabalancebetweenrisksandbenefitstothepatients.Today,itisclearthatweareexperiencingthedawnofanewerainbiomedicalresearchusheredinbygenomeeditingtechnologies. Acknowledgments—WethankHsin-KaiLiaoandYingGuforhelpfuldiscussions;IlirDubova,MaySchwarz,andPeterSchwarzforadministrativesupport;andJamesA.Cooperforhelpwithillustration. REFERENCES
1.Melton,
D.W.(1994)Genetargetinginthemouse.BioEssays16,633–6382.,
C.,andCohen-Tannoudji,
M.(2001)Genomeengineeringvia homologousbinationinmouseembryonicstem(ES)cells:anamazinglyversatiletoolforthestudyofmammalianbiology.An.Acad.Bras.Cienc.73,365–3833.Sancho-Martinez,
I.,Li,
M.,andIzpisuaBelmonte,
J.C.(2011)DiseasecorrectiontheiPSCway:advancesiniPSC-basedtherapy.Clin.Pharmacol.Ther.89,746–7494.Ruby,
K.M.,andZheng,
B.(2009)GenetargetinginaHUESlineofhumanembryonicstemcellsviaelectroporation.StemCells27,1496–15065.Zwaka,
T.P.,andThomson,
J.A.(2003)Homologousbinationinhumanembryonicstemcells.Nat.Biotechnol.21,319–3216.Bañuelos,
C.A.,Banáth,
J.P.,MacPhail,
S.H.,Zhao,
J.,Eaves,
C.A.,O’Connor,
M.D.,Lansdorp,
P.M.,andOlive,
P.L.(2008)Mousebutnothumanembryonicstemcellsaredeficientinrejoiningofionizingradiation-inducedDNAdouble-strandbreaks.DNARepair7,1471–14837.Meek,
K.,Dang,
V.,andLees-Miller,
S.P.(2008)DNA-PK:themeanstojustifytheends?
Adv.Immunol.99,33–588.Cherry,
A.B.,andDaley,
G.Q.(2013)Reprogrammedcellsfordiseasemodelingandregenerativemedicine.Annu.Rev.Med.64,277–2909.Soldner,
F.,andJaenisch,
R.(2012)Medicine.iPSCdiseasemodeling.Science338,1155–115610.Chapman,
J.R.,Taylor,
M.R.,andBoulton,
S.J.(2012)Playingtheendgame:DNAdouble-strandbreakrepairpathwaychoice.Mol.Cell47,497–51011.Carroll,
D.(2011)Genomeengineeringwithzinc-fingernucleases.ics188,773–78212.Bhakta,
M.S.,Henry,
I.M.,Ousterout,
D.G.,Das,
K.T.,Lockwood,
S.H.,Meckler,
J.F.,Wallen,
M.C.,Zykovich,
A.,Yu,
Y.,Leo,
H.,Xu,
L.,Gersbach,
C.A.,andSegal,
D.J.(2013)Highlyactivezinc-fingernucleasesbyextendedmodularassembly.GenomeRes.23,530–53813.Urnov,
F.D.,Rebar,
E.J.,Holmes,
M.C.,Zhang,
H.S.,andGregory,
P.D.(2010)Genomeeditingwithengineeredzincfingernucleases.Nat.Rev..11,636–64614.Haruta,
M.,Tomita,
Y.,Yuno,
A.,Matsumura,
K.,Ikeda,
T.,Takamatsu,
K.,Haga,
E.,Koba,
C.,Nishimura,
Y.,andSenju,
S.(2013)TAP-deficienthumaniPScell-derivedmyeloidcelllinesasunlimitedcellsourcefordendriticcell-likeantigen-presentingcells.GeneTher.20,504–51315.Chang,
C.J.,andBouhassira,
E.E.(2012)Zinc-fingernuclease-mediatedcorrectionof␣-thalassemiainiPScells.Blood120,3906–391416.Wang,
Y.,Zhang,
W.Y.,Hu,
S.,Lan,
F.,Lee,
A.S.,Huber,
B.,Lisowski,
L.,Liang,
P.,Huang,
M.,deAlmeida,
P.E.,Won,
J.H.,Sun,
N.,Robbins,
R.C.,Kay,
M.A.,Urnov,
F.D.,andWu,
J.C.(2012)Genomeeditingofhumanembryonicstemcellsandinducedpluripotentstemcellswithzincfingernucleasesforcellularimaging.Circ.Res.111,1494–150317.Soldner,
F.,Laganière,
J.,Cheng,
A.W.,Hockemeyer,
D.,Gao,
Q.,Alagappan,
R.,Khurana,
V.,Golbe,
L.I.,Myers,
R.H.,Lindquist,
S.,Zhang,
L.,Guschin,
D.,Fong,
L.K.,Vu,
B.J.,Meng,
X.,Urnov,
F.D.,Rebar,
E.J.,Gregory,
P.D.,Zhang,
H.S.,andJaenisch,
R.(2011)GenerationofisogenicpluripotentstemcellsdifferingexclusivelyattwoearlyonsetParkinsonpointmutations.Cell146,318–33118.Collin,
J.,andLako,
M.(2011)Concisereview:puttingafingeronstemcellbiology:zincfingernuclease-driventargetediceditinginhumanpluripotentstemcells.StemCells29,1021–1033 19.Perez-Pinera,
P.,Ousterout,
D.G.,andGersbach,
C.A.(2012)Advancesintargetedgenomeediting.Curr.Opin.Chem.Biol.16,268–277 20.Ramirez,
C.L.,Foley,
J.E.,Wright,
D.A.,Müller-Lerch,
F.,Rahman,
S.H.,Cornu,
T.I.,Winfrey,
R.J.,Sander,
J.D.,Fu,
F.,Townsend,
J.A.,Cathomen,
T.,Voytas,
D.F.,andJoung,
J.K.(2008)Unexpectedfailureratesformodularassemblyofengineeredzincfingers.Nat.Methods5,374–375 21.Gabriel,
R.,Lombardo,
A.,Arens,
A.,Miller,
J.C.,Genovese,
P.,Kaeppel,
C.,Nowrouzi,
A.,Bartholomae,
C.C.,Wang,
J.,Friedman,
G.,Holmes,
M.C.,Gregory,
P.D.,Glimm,
H.,Schmidt,
M.,Naldini,
L.,andvonKalle,
C.(2011)Anunbiasedgenome-wideanalysisofzinc-fingernucleasespecificity.Nat.Biotechnol.29,816–823 22.Doyon,
Y.,Vo,
T.D.,Mendel,
M.C.,Greenberg,
S.G.,Wang,
J.,Xia,
D.F.,Miller,
J.C.,Urnov,
F.D.,Gregory,
P.D.,andHolmes,
M.C.(2011)Enhancingzinc-finger-nucleaseactivitywithimprovedobligateheterodimericarchitectures.Nat.Methods8,74–79 23.Ramirez,
C.L.,Certo,
M.T.,Mussolino,
C.,Goodwin,
M.J.,Cradick,
T.J.,McCaffrey,
A.P.,Cathomen,
T.,Scharenberg,
A.M.,andJoung,
J.K.(2012)Engineeredzincfingernickasesinducehomology-directedrepairwithreducedmutageniceffects.NucleicAcidsRes.40,5560–5568 24.Wang,
J.,Friedman,
G.,Doyon,
Y.,Wang,
N.S.,Li,
C.J.,Miller,
J.C.,Hua,
K.L.,Yan,
J.J.,Babiarz,
J.E.,Gregory,
P.D.,andHolmes,
M.C.(2012)TargetedgeneadditiontoapredeterminedsiteinthehumangenomeusingaZFN-basednickingenzyme.GenomeRes.22,1316–1326 25.Woods,
N.B.,Bottero,
V.,Schmidt,
M.,vonKalle,
C.,andVerma,
I.M.(2006)Genetherapy:therapeuticgenecausinglymphoma.Nature440,1123 26.Pattanayak,
V.,Ramirez,
C.L.,Joung,
J.K.,andLiu,
D.R.(2011)Revealingoff-targetcleavagespecificitiesofzinc-fingernucleasesbyinvitroselection.Nat.Methods8,765–770 27.Yusa,
K.,Rashid,
S.T.,Strick-Marchand,
H.,Varela,
I.,Liu,
P.Q.,Paschon,
D.E.,Miranda,
E.,Ordóñez,
A.,Hannan,
N.R.,Rouhani,
F.J.,Darche,
S.,Alexander,
G.,Marciniak,
S.J.,Fusaki,
N.,Hasegawa,
M.,Holmes,
M.C.,DiSanto,
J.P.,Lomas,
D.A.,Bradley,
A.,andVallier,
L.(2011)Targetedgenecorrectionof␣1-antitrypsindeficiencyininducedpluripotentstemcells.Nature478,391–394 28.Joung,
J.K.,andSander,
J.D.(2013)TALENs:awidelyapplicabletechnologyfortargetedgenomeediting.Nat.Rev.Mol.CellBiol.14,49–55 29.Boch,
J.,Scholze,
H.,Schornack,
S.,Landgraf,
A.,Hahn,
S.,Kay,
S.,Lahaye,
T.,Nickstadt,
A.,andBonas,
U.(2009)BreakingthecodeofDNAbindingspecificityofTAL-typeIIIeffectors.Science326,1509–1512 30.Mak,
A.N.,Bradley,
P.,Cernadas,
R.A.,Bogdanove,
A.J.,andStoddard,
B.L.(2012)ThecrystalstructureofTALeffectorPthXo1boundtoitsDNAtarget.Science335,716–719 31.Reyon,
D.,Tsai,
S.Q.,Khayter,
C.,Foden,
J.A.,Sander,
J.D.,andJoung,
J.K.(2012)FLASHassemblyofTALENsforhigh-throughputgenomeediting.Nat.Biotechnol.30,460–465 32.Schmid-Burgk,
J.L.,Schmidt,
T.,Kaiser,
V.,Höning,
K.,andHornung,
V.(2013)Aligation-independentcloningtechniqueforhigh-throughputassemblyoftranscriptionactivator-likeeffectorgenes.Nat.Biotechnol.31,76–81 33.Briggs,
A.W.,Rios,
X.,Chari,
R.,Yang,
L.,Zhang,
F.,Mali,
P.,andChurch,
G.M.(2012)Iterativecappedassembly:rapidandscalablesynthesisofrepeat-moduleDNAsuchasTALeffectorsfromindividualmonomers.NucleicAcidsRes.40,e117 34.Kim,
Y.,Kweon,
J.,Kim,
A.,Chon,
J.K.,Yoo,
J.Y.,Kim,
H.J.,Kim,
S.,Lee,
C.,Jeong,
E.,Chung,
E.,Kim,
D.,Lee,
M.S.,Go,
E.M.,Song,
H.J.,Kim,
H.,Cho,
N.,Bang,
D.,Kim,
S.,andKim,
J.S.(2013)AlibraryofTALeffectornucleasesspanningthehumangenome.Nat.Biotechnol.31,251–258 35.Mussolino,
C.,Morbitzer,
R.,Lütge,
F.,Dannemann,
N.,Lahaye,
T.,andCathomen,
T.(2011)AnovelTALEnucleasescaffoldenableshighgenomeeditingactivitybinationwithlowtoxicity.NucleicAcidsRes.39,9283–9293 36.Ding,
Q.,Lee,
Y.K.,Schaefer,
E.A.,Peters,
D.T.,Veres,
A.,Kim,
K.,Kuperwasser,
N.,Motola,
D.L.,Meissner,
T.B.,Hendriks,
W.T.,Trevisan,
M.,Gupta,
R.M.,Moisan,
A.,Banks,
E.,Friesen,
M.,Schinzel,
R.T.,Xia,
F.,Tang,
A.,Xia,
Y.,Figueroa,
E.,Wann,
A.,Ahfeldt,
T.,Daheron,
L.,Zhang,
F.,Rubin,
L.L.,Peng,
L.F.,Chung,
R.T.,Musunuru,
K.,andCowan,
C.A.(2013)ATALENgenome-editingsystemforgeneratinghumanstemcell- 4598JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER8•FEBRUARY21,2014 Downloadedfrom/atInstBiophys,CASonJune29,2014 MINIREVIEW:TargetedGenomeEditingTechnologiesinhPSCs baseddiseasemodels.CellStemCell12,238–25137.Moscou,
M.J.,andBogdanove,
A.J.(2009)AsimpleciphergovernsDNA recognitionbyTALeffectors.Science326,150138.Bultmann,
S.,Morbitzer,
R.,Schmidt,
C.S.,Thanisch,
K.,Spada,
F.,El- saesser,
J.,Lahaye,
T.,andLeonhardt,
H.(2012)Targetedtranscriptionalactivationofsilentoct4pluripotencygenebiningdesignerTALEsandinhibitionoficmodifiers.NucleicAcidsRes.40,5368–537739.Valton,
J.,Dupuy,
A.,Daboussi,
F.,Thomas,
S.,Maréchal,
A.,Macmaster,
R.,Melliand,
K.,Juillerat,
A.,andDuchateau,
P.(2012)ingtranscriptionactivator-likeeffector(TALE)DNAbindingdomainsensitivitytocytosinemethylation.J.Biol.Chem.287,38427–3843240.Petek,
L.M.,Russell,
D.W.,andMiller,
D.G.(2010)Frequentendonucleasecleavageatoff-targetlocationsinvivo.Mol.Ther.18,983–98641.Jinek,
M.,Chylinski,
K.,Fonfara,
I.,Hauer,
M.,Doudna,
J.A.,andCharpentier,
E.(2012)Aprogrammabledual-RNA-guidedDNAendonucleaseinadaptivebacterialimmunity.Science337,816–82142.Wiedenheft,
B.,Sternberg,
S.H.,andDoudna,
J.A.(2012)RNA-guidedicsilencingsystemsinbacteriaandarchaea.Nature482,331–33843.Jinek,
M.,East,
A.,Cheng,
A.,Lin,
S.,Ma,
E.,andDoudna,
J.(2013)RNAprogrammedgenomeeditinginhumancells.eLife2,e0047144.Cho,
S.W.,Kim,
S.,Kim,
J.M.,andKim,
J.S.(2013)TargetedgenomeengineeringinhumancellswiththeCas9RNA-guidedendonuclease.Nat.Biotechnol.31,230–23245.Cong,
L.,Ran,
F.A.,Cox,
D.,Lin,
S.,Barretto,
R.,Habib,
N.,Hsu,
P.D.,Wu,
X.,Jiang,
W.,Marraffini,
L.A.,andZhang,
F.(2013)MultiplexgenomeengineeringusingCRISPR/Cassystems.Science339,819–82346.Mali,
P.,Yang,
L.,Esvelt,
K.M.,Aach,
J.,Guell,
M.,DiCarlo,
J.E.,Norville,
J.E.,andChurch,
G.M.(2013)RNA-guidedhumangenomeengineeringviaCas9.Science339,823–82647.Wang,
H.,Yang,
H.,Shivalila,
C.S.,Dawlaty,
M.M.,Cheng,
A.W.,Zhang,
F.,andJaenisch,
R.(2013)One-stepgenerationofmicecarryingmutationsinmultiplegenesbyCRISPR/Cas-mediatedgenomeengineering.Cell153,910–91848.Ding,
Q.,Regan,
S.N.,Xia,
Y.,Oostrom,
L.A.,Cowan,
C.A.,andMusunuru,
K.(2013)EnhancedefficiencyofhumanpluripotentstemcellgenomeeditingthroughreplacingTALENswithCRISPRs.CellStemCell12,393–39449.Fu,
Y.,Foden,
J.A.,Khayter,
C.,Maeder,
M.L.,Reyon,
D.,Joung,
J.K.,andSander,
J.D.(2013)High-frequencyoff-targetmutagenesisinducedbyCRISPR-Casnucleasesinhumancells.Nat.Biotechnol.31,822–82650.Li,
M.,Suzuki,
K.,Qu,
J.,Saini,
P.,Dubova,
I.,Yi,
F.,Lee,
J.,Sancho-Martinez,
I.,Liu,
G.H.,andIzpisuaBelmonte,
J.C.(2011)Efficientcorrectionofhemoglobinopathy-causingmutationsbyhomologousbinationinintegration-freepatientiPSCs.CellRes.21,1740–174451.Liu,
G.H.,Qu,
J.,Suzuki,
K.,Nivet,
E.,Li,
M.,Montserrat,
N.,Yi,
F.,Xu,
X.,Ruiz,
S.,Zhang,
W.,Wagner,
U.,Kim,
A.,Ren,
B.,Li,
Y.,Goebl,
A.,Kim,
J.,Soligalla,
R.D.,Dubova,
I.,Thompson,
J.,Yates,
J.,3rd,Esteban,
C.R.,Sancho-Martinez,
I.,andIzpisuaBelmonte,
J.C.(2012)ProgressivedegenerationofhumanneuralstemcellscausedbypathogenicLRRK2.Nature491,603–607 52.Liu,
G.H.,Suzuki,
K.,Qu,
J.,Sancho-Martinez,
I.,Yi,
F.,Li,
M.,Kumar,
S.,Nivet,
E.,Kim,
J.,Soligalla,
R.D.,Dubova,
I.,Goebl,
A.,Plongthongkum,
N.,Fung,
H.L.,Zhang,
K.,Loring,
J.F.,Laurent,
L.C.,andIzpisuaBelmonte,
J.C.(2011)Targetedgenecorrectionoflaminopathy-associatedLMNAmutationsinpatient-specificiPSCs.CellStemCell8,688–694 53.Suzuki,
K.,Mitsui,
K.,Aizawa,
E.,Hasegawa,
K.,Kawase,
E.,Yamagishi,
T.,Shimizu,
Y.,Suemori,
H.,Nakatsuji,
N.,andMitani,
K.(2008)Highlyefficienttransientgeneexpressionandgenetargetinginprimateembryonicstemcellswithhelper-dependentadenoviralvectors.Proc.Natl.Acad.Sci.U.S.A.105,13781–13786 54.Mitsui,
K.,Suzuki,
K.,Aizawa,
E.,Kawase,
E.,Suemori,
H.,Nakatsuji,
N.,andMitani,
K.(2009)Genetargetinginhumanpluripotentstemcellswithadeno-associatedvirusvectors.Biochem.Biophys.Res.Commun.388,711–717 55.Khan,
I.F.,Hirata,
R.K.,Wang,
P.R.,Li,
Y.,Kho,
J.,Nelson,
A.,Huo,
Y.,Zavaljevski,
M.,Ware,
C.,andRussell,
D.W.(2010)EngineeringofhumanpluripotentstemcellsbyAAV-mediatedgenetargeting.Mol.Ther.18,1192–1199 56.Song,
H.,Chung,
S.K.,andXu,
Y.(2010)ModelingdiseaseinhumanESCsusinganefficientBAC-basedhomologousbinationsystem.CellStemCell6,80–89 57.Alba,
R.,Bosch,
A.,andChillon,
M.(2005)Gutlessadenovirus:last-generationadenovirusforgenetherapy.GeneTher.12,S18–S27 58.Qu,
X.,Wang,
P.,Ding,
D.,Li,
L.,Wang,
H.,Ma,
L.,Zhou,
X.,Liu,
S.,Lin,
S.,Wang,
X.,Zhang,
G.,Liu,
S.,Liu,
L.,Wang,
J.,Zhang,
F.,Lu,
D.,andZhu,
H.(2013)Zinc-finger-nucleasesmediatespecificandefficientexcisionofHIV-1proviralDNAfrominfectedandlatentlyinfectedhumanTcells.NucleicAcidsRes.41,7771–7782 59.Provasi,
E.,Genovese,
P.,Lombardo,
A.,Magnani,
Z.,Liu,
P.Q.,Reik,
A.,Chu,
V.,Paschon,
D.E.,Zhang,
L.,Kuball,
J.,Camisa,
B.,Bondanza,
A.,Casorati,
G.,Ponzoni,
M.,Ciceri,
F.,Bordignon,
C.,Greenberg,
P.D.,Holmes,
M.C.,Gregory,
P.D.,Naldini,
L.,andBonini,
C.(2012)EditingTcellspecificitytowardsleukemiabyzincfingernucleasesandlentiviralgenetransfer.Nat.Med.18,807–815 60.Howden,
S.E.,Gore,
A.,Li,
Z.,Fung,
H.L.,Nisler,
B.S.,Nie,
J.,Chen,
G.,McIntosh,
B.E.,Gulbranson,
D.R.,Diol,
N.R.,Taapken,
S.M.,Vereide,
D.T.,Montgomery,
K.D.,Zhang,
K.,Gamm,
D.M.,andThomson,
J.A.(2011)iccorrectionandanalysisofinducedpluripotentstemcellsfromapatientwithgyrateatrophy.Proc.Natl.Acad.Sci.U.S.A.108,6537–6542 61.Hacein-Bey-Abina,
S.,VonKalle,
C.,Schmidt,
M.,McCormack,
M.P.,Wulffraat,
N.,Leboulch,
P.,Lim,
A.,Osborne,
C.S.,Pawliuk,
R.,Morillon,
E.,Sorensen,
R.,Forster,
A.,Fraser,
P.,Cohen,
J.I.,deSaintBasile,
G.,Alexander,
I.,Wintergerst,
U.,Frebourg,
T.,Aurias,
A.,,
D.,Romana,
S.,Radford-Weiss,
I.,Gross,
F.,Valensi,
F.,Delabesse,
E.,Macintyre,
E.,Sigaux,
F.,Soulier,
J.,Leiva,
L.E.,Wissler,
M.,Prinz,
C.,Rabbitts,
T.H.,LeDeist,
F.,Fischer,
A.,andCavazzana-Calvo,
M.(2003)LMO2-associatedclonalTcellproliferationintwopatientsaftergenetherapyforSCID-X1.Science302,415–419 FEBRUARY21,2014•VOLUME289•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY4599
声明：
该资讯来自于互联网网友发布，如有侵犯您的权益请联系我们。
