edu ABSTRACT Transcription activatorlike effector TALE nucleases TALENs have recently emerged as a revolution ary genome editing tool in many different organisms and cell types The sitespeci64257c chromosomal doublestrand breaks introduced by TALENs ID: 53839
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TranscriptionActivator-LikeEffectorNucleases(TALENs):AHighlyEfÞcientandVersatileToolforGenomeEditingNingSun,HuiminZhaoDepartmentofBiochemistry,UniversityofIllinoisatUrbana-Champaign,600SouthMathewsAvenue,Urbana,Illinois61801 2013WileyPeriodicals,Inc.BiotechnologyandBioengineering,Vol.110,No.7,July,2013 ZFdomainshavelimitedmodularityduetothecontext-dependentDNA-bindingeffects,makingitdifÞcultforZFNstotargetanydesiredDNAsequence(Ramirezetal.,2008).Moreover,lackofspeciÞcityofsomeZFdomainscangenerateoff-targetcleavage,leadingtoundesiredmutationsandchromosomalaberrations(Pattanayaketal.,2011;Radeckeetal.,2010).Recently,transcriptionactivator-likeeffectornucleases(TALENs)haverapidlyemergedasanalternativegenomeeditingtooltoZFNs(recentlyreviewedinJoungandSander,2013).SimilartoZFNs,TALENsusethenon-speciÞcIdomainastheDNAcleavagemoduleandfunctionasdimers(Fig.1B).However,theDNAbindingdomainsofTALENsarecomposedofaseriesoftandemrepeatsasinTALEsoftheplantpathogenicbacteriafromthegenusXanthomonas(asreviewedinBogdanoveandVoytas,2011;MunozBodnaretal.,2013).Eachrepeatcomprises33Ð35aaandrecognizesasinglenucleotide.Thelastrepeattypicallyhasonly20aa,andisthereforecalledaÔÔhalf-repeat.ÕÕTheDNArecognitionspeciÞcityisconferredbythehighlyvariableaminoacidsatpositions12and13(e.g.,NIrecognizesadenine,HDrecognizescytosine,NGrecognizesthymine,andNNrecognizesguanineandadenine)(Bochetal.,2009;MoscouandBogdanove,2009).Unlikethecontext-dependentDNAbindingofZFNs,TALENscanbeeasilyandrapidlyconstructedtotargetalmostanyDNAsequenceduetothesimpleproteinÐDNAcodeandthemodularnature.Inaddition,TALENsexhibitsigniÞcantlyreducedoff-targeteffectsandcytotoxicitiescomparedwithZFNs,makingthemanefÞcientgenomeeditingtool(Dingetal.,2013;Mussolinoetal.,2011).Withinthelast3years,TALENshavebeenwidelyappliedtomodifyendogenousgenesinavarietyoforganisms(TableI).Applicationsincludestudyinggenefunctionsinmodelorganisms,improvingtraitsincropplantsandlivestock,generatingdiseasemodels,andtreatinggeneticdisordersinhumans.Inthisarticle,weprovideacomprehensivereviewofTALENtechnologyincludingtheoptimizationofthescaffold,improvementoftheDNArecognitionspeciÞcity,andassemblyofTALErepeatarrays.DuetotheeaseofdesignandhighefÞciencyofgenomeediting,TALENshaveopenedupmanynewavenuesforbasicandappliedbiologicalresearch.ScaffoldOptimizationTheoriginalTALENconstructionwasreportedbytwoindependentgroups.Lietal.(2011a)fusedthefull-length Figure1.TALEN-mediatedgenomeediting.:Thesite-speciÞcchromosomalDSBisrepairedbyeitherNHEJorHR,resultingingenedisruption,genedeletion,geneinsertionorgenereplacement.:SchematicofTALENarchitecture.ATALENiscomposedofaN-terminalsegment(NTS,pinkbox),acentralrepeatdomain,aC-terminalsegment(CTS,cyanbox),andaIcatalyticdomain(orangeoval).ThecentralrepeatdomaincomprisesaseriesofrepeatunitsthatareresponsibleforspeciÞcrecognitionofthymin(redboxes),adenine(greenboxes),cytosine(blueboxes),andguanine(yellowboxes).TheformationofaheterodimerbytwoTALENsinatail-to-tailorientationatthetargetsiteexecutesasite-speciÞcDNADSB.TheTALEbindingsitesonthetargetDNAareshowninblackandthespacerisshowningray.BiotechnologyandBioengineering,Vol.110,No.7,July,2013 naturalTALE(AvrXa7andPthXo1)withtheIcatalyticdomain,creatingtheTALENsbearinga288aaN-terminalsegment(NTS)anda295aaC-terminalsegment(CTS).Basedonayeastreporterassay,theoptimalspacerlengthbetweenthetwoTALENbindingsiteswasdeterminedtobe16Ð31bp(TableII).Alternatively,theTALENscreatedbyChristianetal.(2010)encompassesa287aaNTSanda231aaCTS.ThisscaffoldallowsefÞcientDNAcleavageagainsttargetsiteswith13Ð30bpspacers(TableII).SincenaturallyoccurringTALEsaretranscriptionactivatorsfromaplantbacterialpathogen,theirNTSsharborproteinsecretionsignalpeptideswhiletheirCTSscontainnuclearlocalizationsignalpeptidesandatranscriptionactivatordomain(Whiteetal.,2009).ThesesequencescanimpairthecatalyticactivitywhenfusedwiththeIcleavagedomain.ToidentifytheoptimumTALENarchitecturewithhighestcleavageefÞciencyandminimalpeptideportion,scaffoldoptimizationhasbeencarriedoutbyseveralgroups.UsingatruncatedNTSwithonly136aa,Milleretal.(2011)constructedaseriesofTALENsbytrimmingtheCTSbetweenthecentralrepeatunitsandtheInucleasedomain.ThisstudyfoundthatdifferentTALENscaffoldspreferdifferentspacerlengthbetweeneachTALENbindingsite.OneTALENscaffoldwasidentiÞedbearinga63aaCTS,whichcoulddriveefÞcientgenemodiÞcationinhumancellswhenseparatedby12Ð20bpspacers(TableII).ThisscaffoldhassincebeenwidelyappliedforefÞcientgenomeeditinginvariousspecies(Cadeetal.,2012;Hockemeyeretal.,2011;Huangetal.,2011;Leietal.,2012;Mooreetal.,2012;Reyonetal.,2012b;Sanderetal.,2011;Tessonetal.,2011;Woodetal.,2011).Inaddition,anotherTALENscaffoldbearingonlya28aaCTSwithanarrowerseparationrangeof12-13bpwasidentiÞed(TableII).Later,Mussolinoetal.(2011)andChristianetal.(2012)reportedTALENswith TableI.ApplicationsofTALENsfortargetedgenomeeditinginvariousorganisms.OrganismsGenesRefs.ArabidopsisthalianaADH1Cermaketal.(2011)BrachypodiumBdABA1Shanetal.(2013)Cattle(BostaurusCarlsonetal.(2012)Cricket(GryllusimaculatusWatanabeetal.(2012)Frog(XenopustropicalisIshibashietal.(2012),Leietal.(2012)Fruitßy(DrosophilamelanogasterLiuetal.(2012)Hamster(CricetulusgriseusCristeaetal.(2013)Human(HomosapiensCermaketal.(2011),Hockemeyeretal.(2011),Kimetal.(2011),Milleretal.(2011),Mussolinoetal.(2011),San-janaetal.(2012),Wangetal.(2012),Reyonetal.(2012b),Sunetal.(2012b),Dingetal.(2013),Schmid-Burgketal.(2013),Stroudetal.(2013)Medaka(OryziaslatipesAnsaietal.(2013)Mouse(MusmusculusSungetal.(2013)Nematode(CaenorhabditiselegansWoodetal.(2011)Rat(RattusnorvegicusTessonetal.(2011),Tongetal.(2012)Rice(OryzasativaL.Lietal.(2012b),Shanetal.(2013)Silkworm(BombyxmoriMaetal.(2012),Sajwanetal.(2013)Swine(SusscrofaCarlsonetal.(2012)Tobacco(NicotianatabacumZhangetal.(2013)Yeast(SaccharomycescerevisiaeLietal.(2011b)ZebraÞsh(DaniorerioHuangetal.(2011),Sanderetal.(2011),Bedelletal.(2012),Cadeetal.(2012),Dahlemetal.(2012),Mooreetal.(2012),Chenetal.(2013)SunandZhao:TranscriptionActivator-LikeEffectorNucleasesBiotechnologyandBioengineering evenshorterCTSsthatexhibitnarrowoptimalrangesofspacers(TableII).AsystematicstudywascarriedoutbySunetal.(2012b)whoconstructed10differentTALENscaffoldswithvariousNTSsandCTSs.TheDNAcleavageactivityofeachscaffoldwasassayedagainst10substrateswithdifferentspacersinayeastreportersystem.Basedonthis10matrix,twoTALENscaffoldswithhighDNAcleavageefÞciencyinbothyeastandhumancellswereidentiÞed.Onebearinga207aaNTSanda31aaCTSpreferstargetsiteswith10Ð16bpspacerswhileanotherbearinga207aaNTSanda63aaCTShashighestefÞciencywhenseparatedby14Ð32bpspacers(TableII).ItisnoteworthythattheTALENwitha50aaNTShasnocatalyticactivityagainstanytargetsites.Lateron,Gaoetal.(2012)solvedthecrystalstructureoftheTALENTSanddiscoveredanextendedN-terminalDNAbindingregioncomposedofthe127aaimmediatelyprecedingthecentralrepeatunits.The127aaNTSfeaturesfourcontinuousrepeats.Eachrepeatcontainstwoandaninterveningloop(Fig.2A),astructuralfeaturehighlysimilartothatofthecentralrepeatunit.Althoughthe127aaNTSdoesnotconfersequencespeciÞcity,itiscrucialforDNAbinding.ThisfeatureexplainswhyalltheeffectiveTALENscaffoldshaveatleast127aaprecedingthecentralrepeatunits(TableII).Asecond-generationGoldyTALENscaffoldhasbeendemonstratedtoimprovethegenomeeditingefÞciencyinzebraÞsh(Bedelletal.,2012).GoldyTALENisbasedonascaffoldpreviouslyreportedbyMilleretal.(2011)whichconsistsofa136aaNTSanda63aaCTS.However,ithasninedifferentaasubstitutionsattheNTSandÞvedifferentaasubstitutionsattheCTS.UsingtheGoldyTALENscaffoldandzebraÞshdeliverysystem,certainlociweremodiÞedwith100%efÞciency.Moreover,theyprovidedtheÞrstexampleofHR-basedgenomeeditinginzebraÞshusingsingle-strandedDNAasadonor.TheGoldyTALENscaffoldwasalsoappliedforefÞcientgeneknockoutinlivestock(Carlsonetal.,2012).BecausetheIcatalyticdomainmustdimerizetobecomeactive,twoTALENsubunitsareassembledasheterodimersatthecleavagesite.However,cleavage-competenthomodimerscomposedofeachsubunitmayalsoformandgenerateoff-targetcleavage,whichcanlimitsafetyorefÞciency.Toaddressthislimitation,obligateheterodimermutationswereintroducedatthedimerinterfaceoftheIcleavagedomainwhichpreventedhomodimerizationbasedonelectrostaticandhydrophobicinteractions.ThecreationofIvariantsthatpreferentiallyheterodimerizesuccessfullyreducedoff-targetcleavageofZFNsandrelievedtoxicity(Doyonetal.,2011;Milleretal.,2007;Szczepeketal.,2007).AsimilarprinciplewasappliedtoTALENsbyCadeetal.(2012)forthegenerationofzebraÞshknockoutlines.TheheterodimericTALENsshowsimilarorevengreateractivitiesthantheirhomodimericcounterparts.Moreover,theTALENsconstructedwithheterodimericIdomainsinducedsmallernumbersofabnormalordeadembryos,indicatingreducedtoxicity.ThisobligateheterodimericTALENconÞgurationhasalsobeenreportedbyothergroupsforgeneknockoutstudies(Dahlemetal.,2012;Huangetal.,2011;Leietal.,2012).DNARecognitionSpeciÞcityTheDNArecognitionspeciÞcityofTALENsisconferredbytherepeat-variablediresidues(RVDs)atpositions12and13ofeachrepeat.Morethan20differentRVDshavebeenidentiÞedinTALEs,amongwhichNI,NG,HD,NN,andHGarethemostcommononesrecognizingthenucleotidesA,T,C,G/A,andT,respectively(Bochetal.,2009;MoscouandBogdanove,2009).Basedoncrystalstructures,TALEbindstotargetDNAasaright-handedsuperhelix(Fig.2B).Eachrepeatunitformsaleft-handed,two-helixbundlethatpresentsanRVD-containinglooptotheDNAmajorgroove(Dengetal.,2012a;Maketal.,2012).TheÞrstresidueofeachRVD(residue12ofeachrepeat),eitherHisorAsn,doesnotcontactDNAdirectly.Instead,thesidechainformsahydrogenbondtothebackbonecarbonyloxygenofAlaatposition8(Ala)ofeachrepeat,stabilizingthelocalconformationoftheRVD-containingloop(Fig.2CÐE).Sequence-speciÞccontactsofTALEstotargetDNAaremadebythesecondresidueofeachRVD(residue13ofeachrepeat)tothecorrespondingbaseonthesensestrand.Inthe TableII.EngineeredTALENscaffoldswithdifferentNTSsandCTSs.NTS(aa)CTS(aa)Spacer(bp)ReportersystemRefs.28829516Ð31-galactosidaseassayinyeastLietal.(2011a)28828516TransientexpressionassayintobaccoleavesMahfouzetal.(2011)28723113Ð30-galactosidaseassayinyeastChristianetal.(2010)2876315MutagenesisinmedakaembryosAnsaietal.(2013)2076314Ð32-galactosidaseassayinyeastSunetal.(2012b)2073110Ð16-galactosidaseassayinyeastSunetal.(2012b)1534712Ð21dsEGFPassayinHEK293Mussolinoetal.(2011)1531712dsEGFPassayinHEK293Mussolinoetal.(2011)1366312Ð20SurveyornucleaseassayinK562Milleretal.(2011)1362812Ð13SurveyornucleaseassayinK562Milleretal.(2011)1361813Ð16-galactosidaseassayinyeastChristianetal.(2012)Thespacerlengthwasnotoptimizedinthestudy.BiotechnologyandBioengineering,Vol.110,No.7,July,2013 HDRVDsspeciÞcforCnucleotides,thecarboxylateoxygenofAspformsahydrogenbondtotheaminegroupofcytosine,whichexcludestheotherbasesthroughphysicalorelectrostaticclash(Fig.2C).InthecaseofNGandHGRVDsspeciÞcforTnucleotides,thebackbonecarbonofthemakesanon-polarvanderWaalscontactwiththemethylgroupoftheopposingthyminebase,whichislessfavorablefortheotherbases(Fig.2DandE).IntheNIRVDsspeciÞcforAnucleotides,thealiphaticsidechainofIlemakesnon-polarvanderWaalsinteractionstoC8andN7ofadenine,whichreducesthebindingafÞnitytotheotherbases(Fig.2F).NNRVDsarecommonlyusedtorecognizeGnucleotides.ThesidechainofAsnresiduemakesahydrogenbondwiththeN7nitrogenoftheopposingguaninebase(Fig.2G).ButsimilarinteractionmightbemadewiththeN7nitrogenofadenine,whichmakesNNRVDsassociatewithAandGnucleotideswithalmostidenticalfrequency.BecauseHDandNNRVDsformhydrogenbondswithDNAbases,thebindingafÞnitiesofHDtocytosineandNNtoguaninearemuchstrongerthanthevanderWaalscontactsofNItoadenineandNG/HGtothymine.Ithasbeensuggestedtoincorporateatleast3Ð4strongRVDsfortheconstructionofefÞcientTALENs(Streubeletal.,2012).ThelackofspeciÞcRVDstorecognizeguaninelimitsTALENsÕbroaderapplicationsbecausenon-speciÞcbindingcangenerateoff-targetcleavage,resultinginunexpectedgenomicinstabilityandcytotoxicity.Morbitzeretal.(2010)discoveredthattheNKRVDscanfacilitatespeciÞctargetingofGnucleotidesthroughinplantafunctionanalysis.Based Figure2.CrystalstructuresofTALEs.:TheTALENTSfeaturesfourcontinuousrepeats,whichareimportantfortheDNAbindingafÞnity(adaptedfromGaoetal.,2012,PDBidentiÞcationcode:4HPZ).:OverallstructureoftheTALEPthXo1centralrepeatdomainincomplexwithitstargetsite(adaptedfromMaketal.,2012,proteindatabank(PDB)identiÞcationcode:3UGM).:InteractionofRVDHDwithcytosine(adaptedfromGaoetal.,2012,PDBidentiÞcationcode:4HPZ).Hydrogenbondsareindicatedbycyanlines.:InteractionofRVDHGwiththymine(adaptedfromMaketal.,2012,PDBidentiÞcationcode:3UGM).Anon-polarvanderWaalsinteractionisshowninadottedline.:InteractionofRVDNGwiththymine(adaptedfromGaoetal.,2012,PDBidentiÞcationcode:4HPZ).:InteractionofRVDNIwithadenine(adaptedfromMaketal.,2012,PDBidentiÞcationcode::InteractionofRVDNNwithguanine(adaptedfromMaketal.,2012,PDBidentiÞcationcode:3UGM).:InteractionofRVDNGwith5-methylcytosine(adaptedfromDengetal.,2012b,PDBidentiÞcationcode:4GJR).:InteractionofRVDNwith5-methylcytosinebasedonastructuralmodel.:InteractionoftheNTSwiththe5-precedingthymine.(AdaptedfromMaketal.,2012,PDBidentiÞcationcode:3UGM.)ThecoloringofthetworepeatsinTALENTSmatchesthatinA.SunandZhao:TranscriptionActivator-LikeEffectorNucleasesBiotechnologyandBioengineering ontheSELEXassay,Milleretal.(2011)providedinvitroevidencethatRVDNKhasamuchstrongerpreferenceforguanineoveradenine,whichrepresentsapromisingcodeforthespeciÞcrecognitionofGnucleotides.However,substitutionofRVDNNwithNKsigniÞcantlyreducedTALENactivityinzebraÞshembryo(Huangetal.,2011).SubstantiallyloweractivitiesinNKcontainingTALEshavealsobeenobservedinplantsandinmammaliancells(Congetal.,2012;Streubeletal.,2012).Therefore,RVDNKisnotidealforguaninerecognitionbecausetheimprovementinspeciÞcitysacriÞcesefÞciency.Alternatively,NHhasbeenreportedasacompetentguanine-speciÞcRVD,whichhasmuchhigherefÞciencythanRVDNK(Congetal.,2012;Streubeletal.,2012).ComputationalmodelinganalysisshowedthattheimidazoleringontheHisoftheNHRVDhasacompactbase-stackinginteractionwiththeguaninebase,suggestingapossiblemechanismforitsincreasedspeciÞcityforGnucleotidewhilemaintainingthebindingafÞnity(Congetal.,2012).Althoughsuccessfullyusedinvariouscellularcontexts,TALEDNAbindingdomainshavebeenreportedtobeincapableoftargetingmethylatedDNA(Bultmannetal.,2012).OftenconsideredastheÞfthbase,5-methylcytosine(5mC)isamajorepigeneticmarkandwidelydistributedinfungi,plant,andmammaliangenomes(Suetal.,2011).Inaddition,5mChasbeenidentiÞedinCpGislandsofmanypromoters,whichareimportantregulatoryregionsforgenomemodiÞcation(Maunakeaetal.,2010).Recently,twogroupsdiscoveredthatRVDNGandN(anasteriskindicatesadeletionatresidue13intherepeatunit)canaccommodate5mCefÞcientlyinvitroandinvivo(Dengetal.,2012b;Valtonetal.,2012).Thymineisstructurallysimilarto5mC,withtheonlydifferenceatposition4,whichisnotinvolvedinbindingtoTALErepeats.ThisobservationindicatesthattheNGRVDspeciÞcforthyminemightbeusedtorecognize5mC.TheproteincrystalstructuresolvedbyDengetal.(2012b)showsthatlackofsidechainofGlyinNGRVDsprovidessufÞcientspacetoaccommodatethe5-methylgroupof5mCandallowstheformationofvanderWaalscontacts(Fig.2H).BecauseRVDsarefollowedimmediatelybytwoconservedGlyresidues,NisroughlyequivalenttoNGexceptforashortenedRVDloop(Fig.2I).UsingNtocodefor5mC,Valtonetal.(2012)demonstratedtheÞrstexampleofTALEN-mediatedmodiÞcationatamethylatedlocusinhumancells.Accommodationof5mCbyTALErepeatsthroughtheRVDNGorNextendstheDNArecognitioncodeandenablesresearcherstodesignTALENstotargethyper-methylatedDNAregions,whichhasgreatpotentialinepigeneticsstudiesandhumantherapeuticapplications.AllnaturallyoccurringTALEtargetsitesareprecededbya-thymineatposition0,whichwaspreviouslybelievedtobeessentialforTALEfunction(Bochetal.,2009;MoscouandBogdanove,2009).TheTALEcrystalstructurerevealsthattwodegeneraterepeatspriortothecentralrepeatdomainappeartocooperatetospecifytheconserved5(Maketal.,2012).TheindoleringofaTrpresidueintherepeatR(-1)formsavanderWaalscontactwiththemethylgroupofthethyminebase,suggestingapossiblemechanismfortheconservedspeciÞcityatposition0(Fig.2J).SunandcoworkersreportedthatTALENswithshorterCTSs(31aa)showhigherefÞciencyagainstnaturalTALErecognitionsitesprecededbya5-TthanthatagainstunnaturalTALEsitesprecededbyA,C,orG.However,TALENvariantswithlongerCTSs(63Ð117aa)arecapableofcleavingunnaturalDNAsubstrateswithsimilarefÞciencycomparedwiththatofnaturalTALEsites(Sunetal.,2012b).Otherstudiesalsoprovidedevidencethatathymineatposition0isnotstrictlyrequiredforTALENactivity(Briggsetal.,2012;Milleretal.,2011;Yuetal.,2011).Notably,therearenineleucinezipper-likeheptadrepeatscloselylinkedtotheCterminusoftheTALEcentralrepeatdomain(YangandGabriel,1995).Theseleucine-richrepeatsmaymediateTALE/DNAinteractionsandincreaseDNAbindingafÞnity,makingthe5-Tlessofarequirement.Detailedstructuralstudiescouldhelpsolvethisuncertainty.Therequirementofapreceding5-TcanbemitigatedusingcertainTALENscaffolds,allowinggreaterßexibilityinchoosingtargetsitesingenomeeditingendeavors.AssemblyofTALERepeatArraysBecauseofthehighsimilaritybetweeneachTALErepeatunit,itischallengingtoconstructplasmidsencodinglongarraysofTALErepeats.Toaddressthislimitation,numerousmethodshavebeendevelopedtoassemblethehighlyrepetitiveTALEcentralrepeatdomainsrapidlyandcost-effectively(JoungandSander,2013).Basedonastandardcloningstrategy,Sanderandcoworkersdescribedarestrictionenzymeandligation(REAL)method,inwhichsingleTALErepeatsarejoinedtogetherusingroutinerestrictiondigestionandligationtechniques(Sanderetal.,2011).Initially,theyconstructedalibraryofplasmidsencodingvariousindividualTALErepeatsbyDNAsynthesis.Intheassemblystep,twoTALErepeatsareÞrstjoinedtogetherbyligatingcompatibleoverhangsgeneratedbydigestionwithrestrictionendonucleases.Next,theligationproductencodingtwoTALErepeatsisjoinedwithanotherTALErepeatdimerinthesamemanner,resultinginaDNAfragmentencodingfourTALErepeats.ThisprocesscontinuesinaniterativefashionuntilaTALErepeatarrayofthedesiredlengthisassembled(Fig.3A).Usingalargeplasmidlibraryofpre-assembledmultipleTALErepeats,REALcanbeperformedinamorerapidandlesslabor-intensivefashion,whichisreferredtoasREAL-Fast(Reyonetal.,2012a).Withthehelpofisocaudamerrestrictionenzymes(e.g.,IandI),aunitassemblymethodhasbeendescribedforbuildinglongTALErepeatarraysinthesimilarhierarchicalfashion(Huangetal.,2011).UtilizationofGoldenGatecloninghasgreatlyfacilitatedandacceleratedthesynthesisofTALEgenes(Cermaketal.,2011;Geissleretal.,2011;Lietal.,2011b,2012a;Morbitzeretal.,2011;Sanjanaetal.,2012;Weberetal.,2011;ZhangBiotechnologyandBioengineering,Vol.110,No.7,July,2013 etal.,2011).GoldenGatecloninghasbeendevelopedtoovercomethedifÞcultyofassemblingthemonomersintoorderedmultimerarrays(Engleretal.,2008).ItemploystypeIISrestrictionendonucleasesthatcutoutsideoftheirrecognitionsequencesandproducenon-palindromic,4bp-overhangs.SincetherecognitionandcleavagesitesarespatiallyseparatedintypeIISrestrictionenzymes,essentiallyanydesired5-overhangsequencecanbegenerated.Moreover,becausethecorrectligationproductslacktheenzymerecognitionsiteandcannotberecut,thecleavageandligationcanbecarriedoutinthesamereactionmixtureinasinglestep.ToconstructaTALEcentralrepeatdomaintorecognizeadesiredtargetsite,eachindividualrepeatmustbeßankedbythetypeIISrestrictionsitesatboth5-and-ends.Restrictionendonucleasedigestionremovestheßankingrestrictionsitesandgeneratesuniqueterminaloverhangs.TheoverhangsaredesignedsothateachrepeatligatesspeciÞcallytoanotherrepeatwithacompatibleoverhang.Thus,the3-endofÞrstrepeatcanonlyligatetothe5-endofthesecondrepeat,the3-endofthesecondrepeatcanonlyligatetothe5-endofthethirdrepeatandsoon.Therefore,thepositionofeachrepeatwithintheTALEcentralrepeatdomainisdeÞnedexclusivelybythegivenoverlap.FortheÞrstroundofGoldenGatereaction,6Ð10repeatsareclonedintoanintermediateplasmid.ThesecondroundofGoldenGatereactionassemblestheTALErepeatarraysofeachintermediateplasmidintotheÞnalbackboneplasmidandmakesthecompleteTALEcentralrepeatdomainfusedwithIorotherfunctionalproteindomains(Fig.3B).ToincreasetheassemblyefÞciency,the Figure3.SchematicofthestrategiesfortheassemblyofTALErepeatarrays.:TheREALstrategybasedonhierarchicalligations(Sanderetal.,2011).:TheGoldenGatecloning-basedstrategy(Cermaketal.,2011).EachrepeatunitisßankedbytherecognitionsitesofatypeIISrestrictionendonuclease(blacktriangles).TheÞrstroundofGoldenGatereactionassemblesmultipleTALErepeatunitsinasinglestep.ThesecondroundofGoldenGatereactionreliesontherecognitionsiteofadifferenttypeIISrestrictionenzyme(pinktriangles),whichassemblesthecompleteTALEgenesfrompre-assembledrepeatmultimers.:TheFLASHassemblymethodbasedonsolid-phaseligation(Reyonetal.,2012b).Ligationsarecarriedoutiterativelyonastreptavidin-coatedmagneticbead(browncircle),whichcontainsanimmobilizedbiotinylatedDNAdouble-strandadaptor(greenbox).TheÞnalligationproductisreleasedfromthesolidphasebyrestrictiondigestionandsubclonedintotheÞnalbackbonevector.SunandZhao:TranscriptionActivator-LikeEffectorNucleasesBiotechnologyandBioengineering andthetoxicgeneareintroducedforblue/whitescreeningandselection,respectively.WiththehelpofpreassembledTALErepeattetramersandtrimers,asingle-stepGoldenGatestrategyhasbeendevelopedtogenerateTALENsthatrecognize15bptargetsiteswithin2days(Dingetal.,2013).Duetoitseaseofuseandpublicavailability,GoldenGateassemblyprovidesaconvenientmeanstoconstructTALENsforacademiclaboratories.Forindustrialscalesynthesis,developmentofasolid-phaseligationstrategyhasfacilitatedthecloningofTALEgenesinahigh-throughputandcost-effectivemanner(Briggsetal.,2012;Reyonetal.,2012b;Wangetal.,2012).Thesolid-phasestrategyassemblesTALErepeatunitsonastreptavidin-coatedmagneticbeadthatcontainsanimmo-bilizedbiotinylatedDNAdouble-strandadaptorwitharestrictionendonucleasesiteononeend.LigationofTALErepeatunitsisunidirectionalanditerative.Ineachcycle,newlyaddedTALErepeatunitsareligatedtotheimmobilizedDNAfragmentsandsubsequentwashingstepsremoveundesiredproducts.Thecycleiscontinueduntilanarrayofthedesiredlengthisassembled.TheÞnalligationproductisthenreleasedfromthesolidphasebyrestrictiondigestionandsubclonedintotheÞnalbackbonevector(Fig.3C).Withthisstrategy,TALErepeatunitsareassembledonsolid-phaseratherthaninsolution,therebyavoidingtheneedforgelisolation,puriÞcation,oranalysisofintermediateplasmids.With376archivedplasmidsencodingTALErepeatsastetramers,trimers,dimers,andmonomers,thefastligation-basedautomatablesolid-phasehigh-throughput(FLASH)systemenablesassemblyof96TALEgenesinlessthanoneday.Withautomation,FLASHcanmakesequence-veriÞedTALEexpressionplasmidsfor$100each,includingthecostoflabor(Reyonetal.,2012b).Insteadofusingapre-assembledplasmidlibrary,iterativecappedassembly(ICA)buildsfull-lengtharraysfromindividualTALErepeatmonomers(Briggsetal.,2012).Introductionofcappingoligonucleotideseliminatesincompleteligationandmonomerself-ligation,whichareessentialfortheproductionofpurefull-lengthTALErepeatarrays.Withautomation,ICAenablesefÞcientassemblyofTALEgenesbearingupto21repeatsfollowedbyligationintoanexpressionplasmidwithin3h.Wangetal.(2012)performedsolid-phaseligationonachip,whichallowsthesynthesisof100TALEgenesbearing16or20repeatsin3days.Recently,aligation-independentcloning(LIC)techniquehasbeendevelopedforhigh-throughputassemblyofTALEgenes(Schmid-Burgketal.,2013).ComparedwithGoldenGatecloning,LICreliesonmuchlonger(10Ð30bp)non-palindromicoverhangstoannealwiththeoverhangsofotherfragmentsinahighlyspeciÞcmanner.Thelongoverhangsaregeneratedbythecontrollable3-exonucleaseactivityofT4DNApolymerase.BecausethefragmentsÕlongoverlapsdonotdissociateduringtransformation,theannealedproductscanbedirectlytransformedintoEscherichiacoliwithoutpriorligationstepandligatedthroughbacterialligases.BecauseofitshighÞdelity,LICcircumventsagar-basedsingle-colonypickingstep,whichallowsgrowthofcellsdirectlyinpolyclonalculturesaftertransformation.Using64repeatdimer-containingplasmids,LICallowsgenerationofcorrectlyassembledTALEgenesbearing18.5repeatunitsinthreedaysthroughahierarchical,twostepassemblyprocess.Inaddition,acomprehensive5-merTALErepeatunitfragmentlibrarycomposedof3,072plasmidswascreated,whichenablesautomatedassemblyof600TALEgenesbearing15.5repeatunitsin1day.ConclusionsandFuturePerspectivesThelast3yearswitnessedthetremendousprogressoftheTALENtechnology.ThescaffoldoptimizationisolatedTALENvariantswithhighDNAcleavageefÞciency,whichisessentialfortargetedgenomeediting.ThecharacterizationofnovelRVDsextendedtheDNArecognitioncodeandhelpedtominimizeoff-targetcleavageactivityofTALENsbyincreasingguaninerecognitionspeciÞcity.DevelopmentofnovelstrategiesforconvenientandquickassemblyofTALErepeatarraysenabledhigh-throughputsynthesisofTALENsandmadeTALENtechnologyaccessibleandaffordableforanyacademicorindustriallab.BesidesTALENs,thereareothertoolsavailableforeditinggenomes.MeganucleasesarenaturalDNAendonucleaseswithhighactivityandspeciÞcity,butitisdifÞculttotailortheirDNArecognitionspeciÞcities.ItisrelativelyeasiertoengineerZFNstotargetcustom-designedDNAsequences,butsomeofthemsufferfromtherequirementofintensivelaborforconstructionandoff-targeteffects(asreviewedinSunetal.,2012a).Recently,Clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)-mediatedDNAcleav-agehasbeenappliedforgenomeediting(Choetal.,2013;Congetal.,2013;Hwangetal.,2013;Jiangetal.,2013;Jineketal.,2013;Malietal.,2013).ThissystemcanbereprogrammedreadilyusingcustomizedRNAsandenablemultiplexgenomeengineering.However,thelimitedtargetspeciÞcity(14bp)cancauseoff-targetcleavageandtherequirementforaprotospaceradjacentmotif(PAM)restrictsitstargetingrange.Comparedwiththesetools,TALENshavetheadvantageofhighspeciÞcityandmodularity,buttherearealsolimitationsthatremaintobeaddressedforfurtherimprovement.ThebulkysizeofTALENsmightlimittheirbroaderapplications,especiallyinthecaseswhenefÞcientgenedeliverycannotbeachieved.DevelopmentofstrategiesforefÞcientdeliveryofTALENgenesintocellswouldenableTALEN-mediatedgenomeeditinginmoredifferentorganismsandcelltypes.Ineukaryoticcells,DNAispackagedintochromatin.Therefore,chromosomalcontextandepigeneticmodiÞca-tionsplayamajorroleintheDNAaccessibilityofTALENs.ThecombinationofepigeneticmodiÞcationtoolsandTALENtechnologycouldexpandtherangeoftargetforTALEN-mediatedgenomemodiÞcations,whichmightbeapotentialareaforfutureexploration.UnlikeZFNs,theoff-BiotechnologyandBioengineering,Vol.110,No.7,July,2013 targeteffectsofTALENshavenotbeencomprehensivelycharacterized.Mussolinoetal.(2011)carriedoutaside-by-sidecomparisonbetweenZFNsandTALENsandfoundsigniÞcantlyreducednuclease-associatedcytotoxicitiesofTALENs.Dingetal.(2013)alsoreportedminimaloff-target-effectsofTALENsusingexomesequencingandwhole-genomesequencingatlowcoverage,buttheystillcouldnotcompletelyruleoutTALENoff-targeteffects.Therefore,carefulscreeningofthecompletegenomeofTALEN-modiÞedcellsusingdeepsequencinganalysiswouldbeinstructiveforsafeuseofTALENs,especiallyforhumanclinicalapplications.OtherthanfusingwithItomakeDNAendonucleases,TALEshavebeenusedtocreatenovelchimericproteinsbyfusingwithotherfunctionalproteindomains.TALE-basedtranscriptionactivatorshavebeenconstructedtoinducetranscriptionofendogenousgenesinplants(Morbitzeretal.,2010)andhumancells(Bultmannetal.,2012;Gargetal.,2012;Geissleretal.,2011;Lietal.,2012c;Tremblayetal.,2012;Zhangetal.,2011).Byfusingwithtranscriptionrepressordomains,TALEshavebeenusedtogenerateartiÞcialrepressorsforsequence-speciÞcgenerepressioninbacteria(Politzetal.,2012),yeast(Blountetal.,2012),plants(Mahfouzetal.,2012),andhumancells(Congetal.,2012;Lietal.,2012c).Inaddition,chimericTALErecombinases(TALERs)havebeenconstructedbyfusingahyperactivatedcatalyticdomainfromtheDNAinvertaseGinwithaTALEcentralrepeatdomain(Merceretal.,2012).TALERswithoptimizedarchitecturerecombineDNAefÞcientlyinbacterialandmammaliancells,providinganalternativeapproachfortargetedgenomeediting.ItwouldbeinterestingtocombineTALEcentralrepeatarrayswithotherdifferentfunctionaldomainsformanydifferentapplicationsinthefuture.Forexample,aTALEcombinedwithaligand-bindingdomaincanbeappliedforhigh-throughputdrugscreening;aTALEcombinedwithaDNAmethyltransferasecanbeusedfortargetedDNAmodiÞca-tion;aTALEcombinedwithahistonedeacetylasecanbeusedforspeciÞcchromatinmodiÞcation;aTALEcombinedwithacytosinedeaminasecanbeappliedforendogenoustargetedmutagenesis,etc.Thankstosimplicityindesign,convenienceinconstruc-tionandhighsuccessratesacrossspecies,TALENtechnologyhasreceivedmuchattentionsinceitsinvention.Althoughchallengesandobstaclesremain,TALENtechnol-ogywillcontinuetobeanimportanttopicforfutureresearchanddevelopmentandbeneÞtbothbasicandappliedbiologicalsciences.WethanktheNationalInstitutesofHealth(R21HL089418)andDepartmentofChemicalandBiomolecularEngineeringattheUni-versityofIllinoisatUrbana-ChampaignforÞnancialsupportinourpreviousandcurrentstudiesonthedevelopmentofnewgenomeengineeringtools.WethankDr.JulienValtonandDr.PhilippeDuchateaufromCellectisS.A.(Paris,France)forkindlysharingthestructuralmodelexhibitingRVDNinteractingwith5mC.AnsaiS,SakumaT,YamamotoT,ArigaH,UemuraN,TakahashiR,KinoshitaM.2013.EfÞcienttargetedmutagenesisinmedakausingcustom-designedtranscriptionactivator-likeeffectornucleases(TALENs).Genetics193(3):739Ð749.BedellVM,WangY,CampbellJM,PoshustaTL,StarkerCG,KrugRG2nd,TanW,PenheiterSG,MaAC,LeungAY,FahrenkrugSC,CarlsonDF,VoytasDF,ClarkKJ,EssnerJJ,EkkerSC.2012.Invivogenomeeditingusingahigh-efÞciencyTALENsystem.Nature491:114Ð118.BlountBA,WeeninkT,VasylechkoS,EllisT.2012.RationaldiversiÞcationofapromoterprovidingÞne-tunedexpressionandorthogonalregula-tionforsyntheticbiology.PLoSONE7:e33279.BochJ,ScholzeH,SchornackS,LandgrafA,HahnS,KayS,LahayeT,NickstadtA,BonasU.2009.BreakingthecodeofDNAbindingspeciÞcityofTAL-typeIIIeffectors.Science326:1509Ð1512.BogdanoveAJ,VoytasDF.2011.TALeffectors:CustomizableproteinsforDNAtargeting.Science333:1843Ð1846.BriggsAW,RiosX,ChariR,YangL,ZhangF,MaliP,ChurchGM.2012.Iterativecappedassembly:Rapidandscalablesynthesisofrepeat-moduleDNAsuchasTALeffectorsfromindividualmonomers.NucleicAcidsRes40:e117.BultmannS,MorbitzerR,SchmidtCS,ThanischK,SpadaF,ElsaesserJ,LahayeT,LeonhardtH.2012.TargetedtranscriptionalactivationofpluripotencygenebycombiningdesignerTALEsandinhibitionofepigeneticmodiÞers.NucleicAcidsRes40:5368Ð5377.CadeL,ReyonD,HwangWY,TsaiSQ,PatelS,KhayterC,JoungJK,SanderJD,PetersonRT,YehJR.2012.HighlyefÞcientgenerationofheritablezebraÞshgenemutationsusinghomo-andheterodimericTALENs.NucleicAcidsRes40(16):8001Ð8010.CarlsonDF,TanW,LillicoSG,StverakovaD,ProudfootC,ChristianM,VoytasDF,LongCR,WhitelawCB,FahrenkrugSC.2012.EfÞcientTALEN-mediatedgeneknockoutinlivestock.ProcNatlAcadSciUSA109:17382Ð17387.CarrollD.2011.Genomeengineeringwithzinc-Þngernucleases.Genetics188(4):773Ð782.CermakT,DoyleEL,ChristianM,WangL,ZhangY,SchmidtC,BallerJA,SomiaNV,BogdanoveAJ,VoytasDF.2011.EfÞcientdesignandassemblyofcustomTALENandotherTALeffector-basedconstructsforDNAtargeting.NucleicAcidsRes39:e82.ChenS,OikonomouG,ChiuCN,NilesBJ,LiuJ,LeeDA,AntoshechkinI,ProberDA.2013.Alarge-scaleinvivoanalysisrevealsthatTALENsaresigniÞcantlymoremutagenicthanZFNsgeneratedusingcontext-dependentassembly.NucleicAcidsRes41:2769Ð2778.ChoSW,KimS,KimJM,KimJS.2013.TargetedgenomeengineeringinhumancellswiththeCas9RNA-guidedendonuclease.NatBiotechnolChristianM,CermakT,DoyleEL,SchmidtC,ZhangF,HummelA,BogdanoveAJ,VoytasDF.2010.TargetingDNAdouble-strandbreakswithTALeffectornucleases.Genetics186:757Ð761.Chri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