moter777tk

REVIEWOpenAccessFunctionalgeneticsforall:engineerednucleases,CRISPRandthegeneeditingrevolutionAnnaFGilles1,2andMichalisAverof1,3*AbstractDevelopmentalbiology,asallexperimentalscience,isempoweredbytechnologicaladvances.
Theavailabilityofgenetictoolsinsomespecies-designatedasmodelorganisms-hasdriventheiruseasmajorplatformsforunderstandingdevelopment,physiologyandbehavior.
Extendingthesetoolstoawiderrangeofspeciesdetermineswhether(andhow)wecanexperimentallyapproachdevelopmentaldiversityandevolution.
Duringthelasttwodecades,comparativedevelopmentalbiology(evo-devo)wasmarkedbytheintroductionofgeneknockdownanddeepsequencingtechnologiesthatareapplicabletoawiderangeofspecies.
Theseapproachesallowedustotestthedevelopmentalroleofspecificgenesindiversespecies,tostudybiologicalprocessesthatarenotaccessibleinestablishedmodelsand,insomecases,toconductgenome-widescreensthatovercomethelimitationsofthecandidategeneapproach.
TherecentdiscoveryofCRISPR/Casasameansofprecisealterationsintothegenomepromisestorevolutionizedevelopmentalgenetics.
Inthisreviewwedescribethedevelopmentofgeneeditingtools,fromzinc-fingernucleasestoTALENsandCRISPR,andexaminetheirapplicationingenetargeting,theirlimitationsandtheopportunitiestheypresentforevo-devo.
Weoutlinetheiruseingeneknock-outandknock-inapproaches,andinmanipulatinggenefunctionsbydirectingmoleculareffectorstospecificsitesinthegenome.
Theease-of-useandefficiencyofCRISPRindiversespeciesprovideanopportunitytoclosethetechnologygapthatexistsbetweenestablishedmodelorganismsandemerginggenetically-tractablespecies.
Keywords:Comparativedevelopmentalbiology,Modelorganisms,Genetargeting,Homologousrecombination,Gene-editingnucleases,CRISPRReviewEvo-devo:drivenbytechnologicaladvancesOurunderstandingofdevelopmentalmechanismsisshapedbytheexperimentalmodelsandapproachesathand.
ThepowerfulgeneticapproachesavailableinDrosophila,C.
elegans,zebrafish,miceandArabidopsishavelargelydrivendevelopmentalresearchduringthepastdecades,focusingitonquestionsthatareexperimentallytractableinthesespecies.
However,biologicaldiversitygreatlysur-passeswhatcanbestudiedintheseorganisms.
Phenom-enasuchasregeneration,polyphenismandchromatindiminutionchallengesomeofourconventionalviewsofdevelopment,butarestillpoorlyunderstoodbecausetheyarenotaccessibleinourcurrentexperimentalmodels.
Also,understandingtheevolutionarypathsbywhichdi-versityisgeneratedrequiresthatwecomparedevelop-mentalmechanismsamongseveralanimals,wellbeyondtheestablishedmodelorganisms.
Exploringthesetopicsrequiresextendingourgeneticapproachestonewspecies.
Establishinggenetictoolsinneworganismshasalwaysbeenachallengeforcomparativedevelopmentalbiology.
Evo-devostartedtoflourishwhencloninggenesandstudyingtheirexpressionpatternsinembryoscouldbeextendedtoawiderangeofanimals,withtheadventofPCRandwhole-mountinsituhybridizationtechniquesinthe1990s.
Thesetechniquesallowed'candidategenes'tobeassociatedwithspecificdevelopmentaleventsindifferentanimalsandforevolutionary-developmentalhypothesestobeformulatedbasedonthisinformation.
Testingthesehypothesesexperimentallyandexploringalternativepossibilitiesinanunbiasedfashionbecame,atthatpoint,majorchallengesforthefutureofevo-devo.
*Correspondence:michalis.
averof@ens-lyon.
fr1InstitutdeGénomiqueFonctionnelledeLyon(IGFL),coleNormaleSupérieuredeLyon,46Alléed'Italie,Lyon69364,FranceFulllistofauthorinformationisavailableattheendofthearticle2014GillesandAverof;licenseeBioMedCentralLtd.
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TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
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0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
GillesandAverofEvoDevo2014,5:43http://www.
evodevojournal.
com/content/5/1/43Twoimportantstepstowardmeetingthosechallengesweremadesincethelate1990s:theestablishmentofgeneknockdownapproachesbasedonRNAiandotherantisensemethods(seebelow)andtheinventionoflow-costdeepsequencingtechnologies,whichopenedthedoortounbiasedgenome-widestudies.
Bothmethodscouldbeappliedtoawiderangeofspecies.
Wewillfocushereonfunctionalgeneticsapproaches.
ThefirstimportantadvanceinthisdirectionwasmadewiththediscoveryofRNAinterference(RNAi),amech-anismthatusessmallRNAs(processedfromlargerdouble-strandedprecursors)torecognizeanddegradespecificRNAtargets[1-4].
RNAiisanaturalmechanismthatevolvedineukaryotestoprotectthegenomeagainstinvadingvirusesandtransposons[4].
ThisdefensemechanismcanberedirectedtotargetspecificmRNAsofinterestbyprovidingdouble-strandedRNAmatch-ingthetargetsequence.
SincetheRNAimachineryisfoundnaturallyinmosteukaryotes,RNAi-mediatedgeneknockdownhasturnedouttobewidelyapplic-able.
ThisapproachhasalsobeencomplementedbyotherantisensemethodsthattargetRNAusingdifferenttypesofoligonucleotides(morpholinos,antagomirs,LNAsandothers[5-8]).
Together,theseantisenseapproacheshavegivenustheopportunitytoknockdowngenefunctionsattheRNAlevelinawiderangeofanimals,includingcnidarians,arthropods,nematodes,planar-ians,annelids,echinoderms,tunicatesandvertebrates(forexample,[1,3,9-13]).
RNAi-basedscreensinnewexperimentalmodelshaveallowedustostudybiologicalproblemsthatweregeneticallyintractableinthepast,suchastissuehomeostasisandregenerationinplanarians[14]orparticularaspectsofinsectphysiologyanddevel-opmentinbeetles(http://ibeetle.
uni-goettingen.
de/).
TheflurryofRNAiandotherantisensestudiescarriedoutattheturnofthecenturyrevealedthepoweroftheseapproaches,butalsotheirlimitations.
Besidestechnicallimitationsrelatingtodelivery,toxicityandoff-targetef-fects,forwhichsolutionsandappropriatecontrolscanoftenbefound[15-17],thereareintrinsiclimitationsinthetypeofgeneticmanipulationthatcanbecarriedout:interferencewithgenefunctionisusuallytransient,unlocalized,andprimarilytargetsmRNA.
Antisenseapproachesdonotusuallyallowustoachievecompletelossofgenefunction,toperformstablegeneticmodifi-cation,topursuegain-of-functionandconditionalap-proaches,ortostudycis-regulatoryelements.
Insomeorganisms,theseknockdownapproacheshavebeencomplementedbytransgenesis[18-24],whichgivesaccesstostablegeneticmodificationandgain-of-functionexperimentsviagenemis-expression.
Transgenesisalsoenablestheuseofreporterconstructstostudycis-regula-toryelementsandtogeneratetoolsforliveimaging,aswellasopportunitiestogeneratemosaicanimals,whereclonesofcellscanbemarked,geneticallymodifiedandcomparedtowild-typecellsinthesameindividual[25].
Thepowerofthetransgenicapproachinnewexperimen-talmodelscanbeseen,forexample,incelllabelingandtracingexperimentscarriedouttostudyregenerativeprogenitorcellsincrustaceansandaxolotls[26,27].
Thedevelopmentoftransgenesisrequiresasignificantinvestmentoftimeandeffort,sothisapproachisstilllimitedtofewspecies.
Amongthefunctionalapproachesthatareapplicabletoawiderangeofspecies,wecanalsocountpharma-cologicaltreatments,whichrelyontheuseofsmallmoleculeeffectorstointerferewithspecificregulatorypathways[28].
Together,thesetechnologicaladvancesallowedevo-devotoadvancefromdescriptivecross-speciescompari-sons(inthe1990s)tocomparisonsofgenefunctionwithinadecade.
Inspiteofthisprogress,mostsystemsarestilllaggingfarbehindstandardmodelsintermsofexperimentalpowerandprecision.
Thearrivalofefficientandwidelyapplicablegeneeditingapproachesissettonarrowthatgap,revolutionizinggeneticapproachesbothinestablishedmodelsandinemergingexperimentalspecies.
GenetargetingapproachesTheabilitytomodifyachosensequenceinitsnativelocusoffersgreatadvantagesoverconventionaltrans-genesisandRNAi-mediatedknockdown,bothintermsofversatilityandprecision.
Itenablesustomanipulatebothcodingsequencesandcis-regulatoryelements,toperformgain-andloss-of-functionexperiments,andtogeneratereportersandsensorsthataccuratelyreflectendogenousgeneexpressionandfunction.
Manipulatingageneinitsnativecontextisalsoamorepreciseap-proachbecauseitallowsustostudygenevariantswithintheirnativecis-regulatoryenvironment,wheretheyareexpressedinbiologicallymeaningfullevelsandpatterns.
ConventionalgenetargetinghasexploitedthenaturalabilityofcellstorecombineDNAfragmentsthatbearhomologoussequences,copyinggeneticchangesfromanengineeredtemplatesequencetoahomologoussiteinthegenome.
Inpracticethisofteninvolvesintegratinganex-ogenoussequence,includingappropriatemarkers,intothelocusofinterest.
Theefficiencyofthisprocessislow,intheorderof1in103-107cellsreceivingthetemplateDNA,anditoccursamongahighbackgroundofnon-homologousin-tegrationevents[29,30].
Forthisreason,conventionalgenetargetingisworkableonlyinsystemswhereweareabletoscreenverylargenumbersoftransfectedcellsandselecttheraretargetingevents,forexample,inculturedmamma-liancellsandinyeast[29,31-33].
Theefficiencyofgenetargeting,however,isstronglyenhanced(100to10,000-fold)whenthetargetedlocusisGillesandAverofEvoDevo2014,5:43Page2of13http://www.
evodevojournal.
com/content/5/1/43disruptedbyadouble-strandDNAbreak[34-38].
Forexample,double-strandbreaksproducedbytheexcisionoftransposableelementsareknowntoinducehomolo-gousrecombinationaroundthesiteofexcision[39,40].
Thus,toimprovetheefficiencyandspecificityofgenetargeting,muchattentionhasfocusedondirectingdouble-strandbreakstouniqueDNAsequencesinthegenome.
Double-strandbreakscanelicittwotypesofmolecularrepairmechanismatthesiteofdamage:non-homologousendjoining(NHEJ)inwhichthebrokenendsarere-ligatedtoeachother,orhomology-directedrepair(HDR)inwhichthebreakisrepairedusingahomologousDNAsequenceastemplate(see[41]).
NHEJandHDRhavedifferentconsequences,whicharebothrelevantforgeneediting.
NHEJisthepredominantrepairmechanism,butitiserror-prone,resultingintheintroductionofsmallin-sertionsordeletions(indels)atthesiteofthebreak.
Thus,NHEJprovidesanefficientwaytodisruptgenefunction(knock-out).
Incontrast,HDRisbasedonprecisecopyingofthetemplateandcanservetoinsertspecificchangesthathavebeenengineeredintherepairtemplate(hom-ology-dependentknock-in).
NHEJcanalsobeusedtoligatethebrokenendstoanexogenouslinearDNAfragment,intheabsenceofsequencehomology(hom-ology-independentknock-in)[42-45].
NHEJandHDRarealmostubiquitousinlivingorganisms,sothesetar-getingapproaches(summarizedinFigure1)couldinprinciplebeappliedinanyspeciesofinterest.
Oneofthefirstapproachesforgeneratingdouble-strandbreaksatspecificsitesinthegenomeexploitednaturalsequence-specificendonucleaseswithlongrec-ognitionsequences.
Theseso-called'meganucleases'recognizesequencesthataretypically15to30nucleo-tideslong,providingsufficientspecificitytotargetuniquesequencesineukaryoticgenomes.
Meganucleaseshavebeensuccessfullyusedingenetargeting[35,36],butengineeringtheseproteinstotargetnewsequenceshasproventobeamajorchallenge[53].
Anotherapproachhasreliedonartificialtriple-helix-formingoligonucleo-tides[54,55],butthescopeofthisapproachisalsolimitedbecausetriple-helixformationisonlypossiblewithsomesequences.
Endonucleaseswithcustomizablesequencespecificitieshavebeenadreamofgenetargetingsincethe1990s.
Modulargeneeditingnucleases:zinc-fingernucleasesandTALENsAmajorbreakthroughcamewiththerealizationthatmodularDNArecognitiondomainscouldbeexploitedcombinatoriallytogeneratenucleasestargetingawiderangeofsequences[56].
Thezinc-fingerdomain,whichtypicallyrecognizes3-nucleotidemotifsonDNA,wasthefirsttobeexploitedinthisway.
Artificialenzymes,calledzinc-fingernucleases(ZFNs),wereengineeredbyjoiningseveralzinc-fingerdomains-recognizingadja-centtrinucleotidemotifs-tothecatalyticdomainoftheendonucleaseFokI(reviewedin[57]).
Sequence-specificitywasfurtherincreasedbyengineeringZFNsinawaythatrequirestheirheterodimerizationthroughtheFokIdomainforefficientcleavage[58].
Thus,targetingan18-nucleotidetargetsitecould(inprinciple)beachievedbyaZFNpaircarrying6zinc-fingerdomainswiththeappropriatesequencespecificities.
Todate,ZFNshavebeenusedtotargetmanygenesindiverseorganisms,exploitingbothNHEJ-mediatedknock-outandHDR-mediatedknock-inapproaches[57].
Despitetheirsuccessindemonstratingthepowerofthemodularapproach,ZFNssuffertwomajordraw-backsthathavelimitedtheiruse.
First,notallsequencescanbetargetedbyZFNs,becausezinc-fingermodulesarenotyetavailableforallpossiblenucleotidetriplets(forexample,[59]).
Second,thesequencespecificityofindividualzinc-fingerdomainscannotalwaysbepre-ciselydefined,andmaybeinfluencedbyneighboringdo-mainsintheprotein[60,61].
ThiscontextdependenceFigure1Genetargetingstrategiesusingtargeteddouble-strandbreaks.
WhenchromosomalDNAiscleaved(redarrowhead),theresultingdouble-strandbreakisrepairedbynon-homologousendjoining(NHEJ)orbyhomology-dependentrepair(HDR).
NHEJmayresultinperfectrejoining,oftheends,orintheintroductionofpointmutationsandindels(knock-out).
NHEJmayalsojoinexogenouslinearDNA(showninyellow)tothebrokenendsofthechromosome(homology-independentknock-in);theorientationandreadingframeintheseinsertionsisrandom,unlessdirectedbycomplementaryoverhangs[42,44,45].
HDRrepairsthedouble-strandbreakbyprecisecopyingofarepairtemplatecarryinganexogenoussequence(showninyellow)flankedbysequenceswithhomologytothetargetedlocus(inblue)(homology-dependentknock-in).
TherepairtemplateusuallyconsistsofcircularplasmidDNAwithlonghomologyarms[46-50]orshortsingle-strandedoligonucleotides(ssODNs)bearing10to40nucleotidesofhomologoussequenceateachend[48,50-52].
GillesandAverofEvoDevo2014,5:43Page3of13http://www.
evodevojournal.
com/content/5/1/43meansthatZFNspecificityisnoteasytopredict,leadingtofailuresintargeting[62]andtheneedforcostlydesignandtesting.
ThemodularapproachwastakenastepfurtherwiththediscoveryoftheTALeffector(TALE)DNA-bindingmodulesofXanthomonasbacteria,andtheirsimpleDNArecognitioncode[63,64].
TALproteinsaretran-scriptionfactorswithamodularDNA-bindingregionthatconsistsofmultipletandemrepeats.
EachoftheserepeatsisasmallDNAbindingdomaincapableofrecognizingasinglenucleotide;twoaminoacidresidueswithineachrepeatdetermineitsspecificityforA,G,CorT,andthisspecificityisnotsignificantlyinfluencedbyneighboringdomains[65].
Thus,usingthesamecombinatoriallogicthatwasappliedtoZFNs,TALef-fectornuclease(TALEN)heterodimerswithpre-definedsequencespecificitiescanbegeneratedbyassemblingmultipleTALrepeats-onepernucleotideofthetargetsite-linkedtothecatalyticdomainofFokI[66-68].
Thus,a24-nucleotidesequencecanbetargetedbygeneratingaTALENheterodimer,whereeachmonomerconsistsofanarrayof12TALrepeatsfusedtoFokI.
TALENsofferthreegreatadvantagesoverZFNs.
First,themodularityoftheTALdomainsandthesimplicityoftheirDNArecognitioncodemeanthatvirtuallyanysequencecanbetargetedbyTALENs.
Second,thespecificityofTALdomainsdoesnotappeartobeascontext-dependentasthatofzincfingers,whichre-sultsinmoreaccuratepredictionsoftargetspecificityandahighertargetingsuccessrate.
Third,genetargetingexperimentsindiversespeciesrevealthatTALENsareveryefficient,yieldingtargetingefficienciesashighas30to100%forNHEJ-mediatedknock-outsand1to10%forHDR-mediatedknock-ins(measuredasthefractionofinjectedindividualsgivingrisetoprogenycarryingatargetedallele).
Hightargetingrateshavebeenachievedinawiderangeoforganisms,includinginsects,nematodes,annelids,tunicates,vertebratesanddiverseplants[69-76].
ThefactthateachTALdomaintargetsasinglenucleo-tidemeansthatlongTALarraysneedtobeassembledinordertotargetuniquesequencesinaeukaryoticgen-ome.
Ingeniousprotocolshavebeendevelopedforthispurpose[77-79],bringingTALENtechnologywithinthereachofeverycompetentmolecularbiologylab.
SimpleandefficientgeneeditingusingtheRNA-guidednucleaseCRISPR/Cas9Thelasttwoyearshaveseenthedevelopmentofanewapproachtobuildendonucleaseswithcustomizedsequencespecificities,whichhasrevolutionizedgeneeditingbyitssimplicityandefficiency.
Theapproachisborrowedfromahighlyefficientimmunemechanismofbacteriaandarchaea,whichemploysRNA-guidedendonucleasestospecificallytargetanddegradeviralDNA[80-82](reviewedin[83]).
Thegenomesofmanyprokaryotespossesshypervari-ableloci,termedclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR),whichincorporateshortsequencesfrominvadingvirusesandexpressthemintheformofCRISPR-derivedRNAs(crRNAs).
ThesesmallRNAsassociatewithspecificCRISPR-associated(Cas)proteinstoformanactiveCRISPR/Casendonucle-asecomplex,whosespecificityisdeterminedbysimplebasecomplementaritybetweencrRNAandthetargetviralDNA.
ImmunitytoaviralinfectionisdeterminedbythepresenceofcorrespondingviralsequencesinCRISPRloci[80,84,85].
TheCRISPRmechanismbearssomestrikinganalogieswitheukaryoticRNAiandpiRNA-mediateddefensemechanismsa[83,86].
Inaground-breakingstudypublishedin2012,Jinekandcolleaguesdemonstratedthatthisnucleotide-basedrecogni-tionmechanismcouldprovideastraightforwardapproachforgeneratingcustomizablenucleasesforgenetargeting[87].
TheyusedtheCRISPRsystemofStreptococcuspyogenes,whichinvolvesasingleCasprotein(Cas9)andtwoRNAs(crRNAandtrans-actingantisenseRNA,alsoknownastracRNA)tobuildanactiveCRISPR/Casendonucleasecomplex.
Jineketal.
showedthatitispossibletocombinethesetwoRNAsintoasinglechimericguideRNA(knownasgRNAorsgRNA)thatcanefficientlydirectCas9activitytospecificDNAtargetsinvitro(Figure2).
TheguideRNAhasaregionof20nucleotidesatits5′end,whichbindsthetargetDNAanddeterminesspecificity;any20-nucleotidesequence(N20)canbeplacedatthatsiteb.
The3′regionoftheguideRNA,correspondingtothebacterialtracRNA,isanin-variablesequencethatisrequiredtoformacomplexwithCas9.
Targetrecognitionalsodependsonadditionalinterac-tionsbetweenCas9andthetargetDNA,whichrequirethepresenceofaspecificsequencemotif,the'protospa-ceradjacentmotif'(PAM),immediatelydownstreamofthe20-nucleotidesequencetargetedbytheguideRNA.
ThePAMsequencedoesnothaveacounterpartontheguideRNA(Figure2).
StreptococcuspyogenesCRISPR/Cas9requiresaPAMthatisNGG;itcanthustargetanysequencethatmatchesthemotifN20NGG.
Onceatargetisbound,twoseparatenucleasedomainsofCas9arein-volvedincleavingeachstrandofthetargetDNA.
Cleav-ageoccurswithintheguideRNAtargetregion,usuallythreenucleotidesupstreamofthePAM[81,82,87].
Withinlessthantwoyearssincethefirstdemonstra-tionofCRISPR-mediatedgeneediting[89-93],therehasbeenanexplosionofreportsdescribingtheapplicationofCRISPRindiverseanimalandplantspecies(reviewedin[94]).
Theapproachestogenerateknock-outsandknock-insaresimilartothosepreviouslydescribedforZFNsandTALENs,relyingontheendonucleasetogen-erateadouble-strandbreakatthetargetedlocusandonGillesandAverofEvoDevo2014,5:43Page4of13http://www.
evodevojournal.
com/content/5/1/43thecell'simpreciseortemplate-directedmechanismsofrepairingthatbreak(Figure1).
However,comparedtoZFNsandTALENs,CRISPRhasradicallyimprovedtheaccessibilityofgenetargetingduetoitsstraightforwardapproachforcustomizingsequencespecificity,viatarget-specificguideRNAs.
Itstargetingefficienciesarecompar-ablewiththebestefficienciesachievedusingTALENsinawiderangeofanimalsandplants(forexample,[46,51,95,96]),includingorganismswheregenetarget-ingisnotyetwidelyavailable,suchassilkmoths,axo-lotls,Xenopusandmonkeys[97-100].
Moreover,whileTALENactivityisinhibitedbyDNAmethylation[101],CRISPRactivitydoesnotappeartobeso[102].
Table1summarizestherelativebenefitsanddrawbacksofZFNs,TALENsandCRISPR.
CRISPRdeliveryandtargetrangeDifferentapproacheshavebeenusedtodelivergene-editingnucleasesintotargetcells,includingmicroinjec-tionandtransfection.
CRISPRsystemsrequirethedeliveryofCas9togetherwithguideRNA.
Cas9maybeexpressedfromahelperplasmidcarryingthecodingsequenceofCas9(fusedwithanuclearlocalizationsignalandsometimes'codon-optimized')underthecontrolofanappropriatepromoter.
Alternatively,ifapromoterisunavailable,Cas9canbeprovidedintheformofinvitrotranscribedcappedmRNAoraspurifiedre-combinantprotein[104,105].
Inestablishedmodels,suchasDrosophila,transgenicstrainshavebeengener-atedthatexpressCas9inthegermline[47,48,96].
DeliveryoftheguideRNAismoreconstrainedbe-causeinvitrotranscriptionorplasmid-derivedexpres-sionimposesomelimitationsonthesequenceoftheRNAandmay,therefore,influencetherangeofpotentialtargets.
InvitrotranscriptionofRNAisusuallycarriedoutusingtheRNApolymeraseofbacteriophagesT7,T3orSP6,whichgeneratetranscriptsthatstartwithGG(T3orT7RNApolymerase)orGA(SP6polymerase)[106].
ThealternativetoinvitrosynthesisistoexpresstheguideRNAfromaplasmidortransgeneinvivo.
SmallRNAsareconventionallyexpressedusingRNApolymeraseIIIpromoters,becausetheyoftenrequirepreciselydefinedinitiationandterminationsitesandshouldnotenterthemRNAprocessingpathway.
Thus,guideRNAsareusuallyexpressedviatheU6snRNATable1ComparisonofZFN,TALENandCRISPRapproachesZFNTALENCRISPRModeofactionDNAbreaktargetedbyprotein-DNArecognitionDNAbreaktargetedbyprotein-DNArecognitionDNAbreaktargetedbyRNA-DNAbasecomplementarityNucleasedesignandassemblyDifficult(commercialservicesexpensive)FeasibleinmostlabsbutlaborintensiveEasy(seeTable2)SuccessrateofnucleasedesignaLowHighHighTargetingefficiencyVariableHighwithmostnucleasesHighwithmostguideRNAsTargetrangeLimitedbyrangeandcontext-dependenceofZFmodulesUnlimitedLimitedbyPAMsequence(potentiallyunlimited)Potentialoff-targeteffectsYesYesYesSensitivitytoDNAmethylationNotknownSensitivetoCpGmethylationNotsensitivetoCpGmethylationHighthroughputtargetingNoLimitedYesasee[71,103].
PAM,protospaceradjacentmotif;TALEN,TALeffectornuclease;ZFN,zinc-fingernuclease.
Figure2CRISPR/Cas9interactingwithtargetDNA.
TheCRISPR/Cas9complexofStreptococcuspyogenesconsistsoftheCas9protein(ingray)andaguideRNAthatisachimeraofnaturalcrRNAandtracRNA(inorange).
Thetargetingsequenceatthe5′endoftheguideRNAbase-pairswithcomplementarysequencesonthetargetDNA(inblue);thetargetingsequenceis20nucleotideslong,butmaybeshortenedtoincreasespecificity[88](theadditionof1to2unpairednucleotidesatthe5′endisalsotolerated[51,88]).
ThepresenceofaPAM(protospaceradjacentmotif,NGGforStreptococcuspyogenes),locatedimmediatelydownstreamofthe20-nucleotidesequencetargetedbytheguideRNA,isalsoessentialfortargetrecognitionandcleavage.
ThePAMsequencedoesnothaveacounterpartontheguideRNA.
FollowingrecognitionofthePAMandbase-pairingbetweentheguideRNAandthetarget,Cas9cleaveseachofthetargetDNAstrandsafewnucleotidesupstreamofthePAM(redarrowheads).
EachstrandiscleavedbyadifferentnucleasedomainpresentinCas9(HNHandRuvCdomains).
ThesedomainshavebeenmutatedindependentlytogenerateCas9nickases[82,87].
GillesandAverofEvoDevo2014,5:43Page5of13http://www.
evodevojournal.
com/content/5/1/43promoter[46,89-91,107];U6promotersgeneratetran-scriptsthatstartwithaG.
Inprinciple,thesesequenceconstraintsdictatethat,usingStreptococcuspyogenesCas9(withNGGasaPAM),wecanoptimallytargetsequencesthatcontainGGN18NGGorGAN18NGGmotifsusinginvitrotran-scribedguideRNAsandGN19NGGusingtheU6pro-motertodriveguideRNAexpression.
Inpractice,however,itseemsthatmismatchesatthe5′endoftheguideRNAarewelltolerated,givingacceptablelevelsofgenetargeting[51,88].
Moreover,alternativeguideRNAexpressionstrategiesareemerging,whichovercometheconstraintsimposedbytheU6promoter[108-110].
Thus,thePAMsequencemaybetheonlystringentlimi-tationtoCRISPR'stargetrange.
PAMrecognitionseemstoplayakeyroleinCRISPRtargetrecognition[111],sotherequirementforaPAMinthetargetsequenceislikelytoremain.
These-quenceconstraintsimposedbythePAMmaybeover-comebyexploitingthenaturaldiversityofCRISPRsystems[112-114],orbyrationaldesignandartificialselectionofCas9variantsthatrecognizedifferentPAMsequences.
Off-targeteffectsWhetherusingZFNs,TALENsorCRISPR,targetingachosen,uniquesequenceinthegenomemaybeaccom-paniedbyunintendedcleavageatotherloci.
Severalstudieshaveinvestigatedthespecificityandpotentialoff-targetsofCRISPR[89,102,115-121],focusingonthestringencyofbase-pairingbetweentheguideRNAandthetarget.
Thesestudieshaveestablishedthatmis-matchesaretolerated,especiallyatthe5′endoftheguideRNA,butthattherearenosimplerulespredictingthelikelihoodofmis-targetingbasedonthenumberandpositionofmismatches.
Insomecases,evensequenceswithmultiplemismatchestotheguideRNAweretar-getedefficiently[116].
Inonestudy,targetingspecificitydeterioratedwhenhighconcentrationsofCRISPR/Cas9wereused[118].
ArecentstudyhasalsohighlightedtheroleplayedbythePAMsequenceasCRISPR/Cas9interrogatescom-plexDNAsequencestoidentifytargetsites[111].
ThestudyshowsthattheCRISPR/Cas9complexfirstidenti-fiespotentialtargetsbasedonthePAMsequence,andtheninterrogatestheseforsequencecomplementaritywiththeguideRNA.
ThecomplexdoesnotappeartointeractwithsequencesthatmatchtheguideRNAtargetingmotif(N20)buthavenoadjacentPAM.
Theseobservationssuggestthatoff-targetswillgenerallynotincludesequencesthatarelackingthePAM.
Inspecieswherethegenomesequenceisknown,computationaltoolsarenowroutinelyusedtoselectguideRNAsandtoevaluatepotentialoff-targetingbasedonsequencesimilarityandonthepresenceofaPAM(seeOnlineResourcesforCRISPRinTable2).
Anumberofapproachescanbetakentoconfronttheoff-targetproblemandtomitigateitseffects.
First,itisoftenpossibletocontrolforunspecificeffectsthroughappropriateexperimentaldesign.
AstrategycommonlyemployedinRNAistudiesistoexaminewhethercon-sistentphenotypesareobtainedbytargetingdifferentpartsofagene,usingnon-overlappingdouble-strandedRNAfragmentsorsiRNAs[17].
ThesamestrategycanbeeasilyappliedinmostcasesofgenetargetingbyCRISPR,byusingdifferentguideRNAs.
GuideRNAstargetingdifferentsequencesareveryunlikelytosharethesameoff-targeteffects.
Notably,heteroalleliccombi-nationsofknock-outsgeneratedusingdifferentguideRNAsarelikelytocomplementoff-targetmutationsandtogivehighlyspecificknock-outphenotypes.
StrategiesforimprovingthespecificityofCRISPRarealsobeginningtoemerge,exploitingthecombinedac-tionofpairsofCRISPRnucleases,ormethodsthatin-creasethespecificityofindividualnucleases.
ThefirstapproachreliesonmutantsofCas9,knownasnickases,thatcleaveonlyoneofthetwoDNAstrands.
Usingsuchmutants,adoublestrandbreakcanbegeneratedbytarget-ingapairofcloselylinkedsingle-strandbreaks(nicks)onoppositeDNAstrands.
Therequirementthatthesenickscoincidedrasticallyimprovestargetingspecificitycom-paredtothatofsingleCRISPRnucleases[44,117,119,124].
AvariantofthisapproachcombinestheCRISPR/Cas9DNAbindingactivitywiththeFokIendonuclease,whosedimerizationrequirementsensurethatnonickingoccursatoff-targetsites[109,125].
Forefficientcleavage,these'pairednickase'approachesrequirethatadjacenttargetsites,offsetbyupto30nucleotides,canbefoundonoppositeDNAstrands.
AsecondapproachtoincreasethespecificityoftargetingbyCRISPRreliesontheobservationthatshortrecognitionsequencesarelessforgivingintermsofallowedmismatchesbetweentheguideRNAanditstargets[88](similarobser-vationsonspecificityandtargetsizehavebeenmadewithTALENs,[65]).
Thus,guideRNAswithtargetingsequencesof17to19nucleotidesshowhightargetingefficienciesandmuchreducedoff-targeteffectscomparedtooneswithcanonical20-nucleotidetargetingsequences[88].
Ultimately,itmayalsobepossibletoimprovethestringencyofCRISPRtargetrecognitionbyselectingorengineeringCas9nucleasesthatareintrinsicallylesspromiscuous.
Opportunitiesforevo-devoandfuturechallengesCRISPR-mediatedgenetargetingopensawiderangeofopportunities,bothinestablishedmodelorganismsandinnewlyemergingones.
AquickguideforapplyingCRISPRinnewspeciesisgiveninTable2.
GillesandAverofEvoDevo2014,5:43Page6of13http://www.
evodevojournal.
com/content/5/1/43Table2AquickguidetoCRISPRforbeginnersa1.
PrerequisitesDeliverymethod,reachingthegermlineorothercellsofinterest:microinjection,transfection,electroporationGenomicsequenceoftargetgenesRobustphenotypicassaystodeterminetheeffectofgenetargeting2.
ExperimentalstrategyDecideonthetargetingapproach(knock-inorknock-out),dependingonwhetheryouwanttodisruptgenefunction,engineeraspecificmutation,generateareporter,etcetera.
WhentestingCRISPRforthefirsttime,chooseasimpleknock-outapproach,selectingtargetsthatproducephenotypesthatareeasytoscore,suchaspigmentationgenesoraGFPtransgene[70,98,122],orgeneswithaknown,robustandspecificphenotype.
Forknock-insToknock-inlargeconstructs,useHDRtemplatesinwhichtheknock-inconstructisflankedbyhomologyarms-typically>1kbinlength-matchingthesequencesoneithersideofthedouble-strandbreak[46-49];shorterhomologyarmsgivelowerefficiencies[50].
Providethetemplateasacircularplasmid.
Tointroducesmallchanges,usesyntheticsingle-strandedoligos(ssODNs)bearing10to40nucleotidesofhomologoussequenceateachendastemplatesforHDR[48,50-52].
ThesequencetargetedbyCRISPRshouldbemutatedintherepairtemplatetoprotectthetemplateandtargetedallelesfromcleavage.
Alternatively,ahomology-independentknock-inapproach(seeFigure1)maybeusedtoknock-inlargeDNAfragments[42,45]orshortdouble-strandedoligos(dsODNs)[44].
Usingthisapproach,theinsertionmaybeimprecise[45]ordirectedbycomplementaryoverhangs[42,44].
SelectanapproachthatwillminimizelethalityduetoNHEJ-mediatedindelsinsomatictissues,forexample,byrestrictingCRISPR/Casactivitytothegermline[48,123],targetingconstructstointrons,oradoptingastrategythatimprovestherelativeefficiencyofknock-ins[42,45,50].
3.
DesignofguideRNAs-findingtargetsequencesUsethemostreliablegenomicsequenceavailableforthetargetgene.
Considerthatthetargetedsitemaybearnucleotidepolymorphisms;ifthisislikelytobeanissue,obtainsequencesfromthestrainusedforgenetargetingand/ortestmultipleguideRNAs.
Useonlinesoftwaretosearchforpotentialtargetsites(seeOnlineResourcesforCRISPR,below).
ThesoftwaresearchagivensequenceforsiteswithasuitablePAMmotif(NGGforS.
pyogenesCas9)andadditionalsequenceconstraintsdependingonthemodeofguideRNAproduction(GGN18NGGforinvitroT7-synthesisofguideRNAs,GN19NGGforU6-mediatedexpression).
Thelatterrequirementscanberelaxed,asextraGsmaybeaddedtothe5′endoftheguideRNAwithoutsignificantlycompromisingtargetingefficiency[51,88].
Whenworkingwithasequencedgenome,thesoftwarecanalsodetectpotentialunintendedtargetsandhelpselectguideRNAswithfeweroff-targets.
AlthoughthepresenceofthePAMsequenceatthegenomictargetsiteisessential,itshouldnotbeincludedintheguideRNA(seeFigure2).
ForanN20NGGtargetsite,onlytheN20sequenceisincorporatedatthe5′endoftheguideRNA.
DesignandtestmultipleguideRNAs,ifpossible,tocontrolforoff-targeteffectsandbecausesomeguideRNAsfail(duetopolymorphisms,RNAsecondarystructureorforunexpectedreasons).
Strategiestoreduceoff-targeteffectsmayrequirespecialdesignofguideRNAs:pairednickaseapproachesrequirepairsoftargetsequencesoffsetbynomorethat30nucleotidesonoppositeDNAstrands[44,109,117,119,124,125];truncatedguideRNAsbeartargetingsequencesthatareshorterthan20nucleotides[88].
4.
ProvidingguideRNAsandCas9GuideRNAsareeasilygeneratedbycloningpairsofsyntheticoligos,correspondingtothetwostrandsofthetargetsequence(determinedabove),intovectorscarryingtheinvariableportionoftheguideRNA(availableatwww.
addgene.
org/CRISPR).
Cloningisfacilitatedbyarestrictionsiteonthevector-usuallyBbsIorBsaI,whichdoesnotconstraintheclonedsequence-andcompatibleoverhangsintheannealedoligos.
TheguideRNAscanbeexpressedeitherbyinvitrotranscriptionviathebacteriophageT3,T7orSP6promoters,orbyinvivoexpressionviatheU6promoter.
ForinitialexperimentsinspecieswhereU6promotersandterminatorsareuntested,chooseinvitrosynthesisoftheguideRNA.
Vectorsandprotocolscanbefoundatwww.
addgene.
org/CRISPR.
Cas9canbeexpressedfromahelperplasmidcarryingthecodingsequenceofCas9underthecontrolofanappropriatepromoter.
Alternatively,ifapromoterisunavailableforthespeciesofinterest,Cas9canbeprovidedintheformofinvitrotranscribedcappedmRNAoraspurifiedrecombinantprotein[104,105].
Forinitialexperimentsperformedbymicroinjection,theuseofrecombinantCas9proteinovercomesuncertaintieswithuntestedpromotersandmRNAtranslation.
5.
RapidassaysofCRISPRactivityandgenotypingThemeltingcurveandsurveyororT7E1endonucleaseassaysareinvaluableforarapidassessmentofCRISPRactivityinnewspecies,forroutinetestingofnewguideRNAspriortomorelaboriousexperiments,andforgenotypinganimalsatspecifictargetsites.
TheseassaysdetectindelsandotherpointmutationsgeneratedbyNHEJ.
TheyrelyonPCRandrequireonlyasmallamountofstartingmaterial.
GenomicDNAisextractedfromembryosortissuestobetestedandPCRisperformedusingprimersthatflankthetargetsite.
UntreatedgenomicDNAgivesaPCRproductwithperfectlyannealedstrands(unlesstherearenaturalpolymorphismswithinthefragment),whereasmutagenizedgenomicDNAalsoyieldssomeheteroduplexDNA,consistingofstrandsthatdifferbysmallindelsandpointmutations.
Thefollowingassaysareusedtodetectofthesemismatches.
GillesandAverofEvoDevo2014,5:43Page7of13http://www.
evodevojournal.
com/content/5/1/43Knock-outapproacheswillsurelybemorewidelyappliedinnewlyestablishedexperimentalsystemsduetotheextraordinaryefficiencyofNHEJ-mediatedknock-out,whichapproaches100%insomespecies.
Generatinganull-alleleordisruptingaspecificcis-regulatoryelem-entnowseemswithinreachinawiderangeofanimalsandwillbeprimarilylimitedbyourabilitytoscreenforthesemutations(byphenotypeorbyPCR)andtomain-tainmutantstocks.
Tosomeextent,thehighefficiencyofCRISPR-andTALEN-mediatedknock-outmayalsohelptoovercometheproblemofstock-keeping.
Thehighfrequencyofbi-allelicknock-outininjectedembryosusingCRISPRorTALENhasraisedthepossibilityofcarryingout'G0Table2AquickguidetoCRISPRforbeginnersa(Continued)-Surveyor/T7E1endonucleaseassaysarebasedoncleavageoftheheteroduplexesbyamismatch-specificendonuclease-eitherSurveyororT7endonuclease1[126,127].
Cleavageproducts,indicatingthepresenceofmispairedDNA,aredetectedbyelectrophoresisonanagarosegel.
Thisisasensitivedetectionmethod,bestperformedon400to800bpampliconswithtargetsitespositionednearthemiddle.
-Themeltingcurveassay[128]reliesonthefactthatheteroduplexDNAhasalowermeltingtemperaturethanthecorrespondinghomoduplexfragments.
Thattemperaturedifference,whichisintheorderof1to2°Cfor100to200bpfragments,canbedetectedbyperformingmeltingcurvesinreal-timePCRinstrumentswithhightemperatureresolution.
MorespecificPCR-basedassayscanbedevisedforknock-inapproaches,employingpairsofprimersthatspanthegenomiclocusandknock-infragment.
ThePCRproductscanbeclonedandsequencedtoexaminethenatureandspectrumofcorrespondingmutations.
6.
ScoringphenotypesTheeffectsofCRISPRtargetingcanbeassessedintheanimalswhereCRISPRwasdelivered(G0)orintheirprogeny.
ItisimportanttokeepinmindthatG0saremosaicswhereonlysomecellsarelikelytocarryallelestargetedbyCRISPR;inthebestcasesasignificantproportionoftheanimalshowsbi-allelictargetingandacorrespondingphenotype.
Thedegreeanddistributionoftargetedcellcloneshoweveraredifficulttodetermine,unlessacell-autonomousmarkerisused(forexample,targetingofsomepigmentgenes,knock-inofGFP).
IfthegermlineofG0shasbeenhit,targetedalleleswillberecoveredinthenextgeneration(G1).
IncontrasttoG0s,G1individualsarenon-mosaicandmayinheritonetargetedallele(perlocus)fromtheCRISPR-targetedparent.
AnimalsmaybegenotypedbyPCR(seeabove)andcrossedtoproducehomozygotesandtomaintainmutantlines.
Choosingreliable,specificphenotypicassaysandappropriatecontrolsiscrucial.
Phenotypesmaybesubtleorshowincompletepenetrance.
7.
Off-targeteffectsandcontrolsUnintendedtargets(off-targets)maybeanywhereinthegenomeandaredifficulttopredict.
Twostrategiescanhelptoovercomeproblemswithoff-targeteffects:appropriateexperimentaldesignallowingustodetectandaccountforoff-targeteffectsandapproachesthatimprovethespecificityofCRISPR.
Inmostcasesitispossibletocontrolforoff-targetsbyusingdifferentguideRNAstoachievetargeting;guideRNAstargetingdifferentsequencesareveryunlikelytosharethesameoff-targets.
AllelesgeneratedusingdifferentguideRNAsmaybebroughttogetherbycrossing,inheteroalleliccombina-tionsthatarelikelytocomplementoff-targetmutations.
ThespecificityofCRISPRcanbesignificantlyimprovedbyusingpairednickases[44,109,117,119,124,125]ortruncatedguideRNAs[88](seemaintext).
Off-targetmutationswillsegregateawayfromtargetedallelesingeneticcrosses,unlesstheyarelinkedonthechromosome.
8.
OnlineresourcesforCRISPRGeneralwww.
genome-engineering.
org/crisprwww.
addgene.
org/CRISPRwww.
flycrispr.
molbio.
wisc.
eduwww.
crisprflydesign.
orggroups.
google.
com/forum/#!
forum/crisprSoftwarefordesigningguideRNAscrispr.
mit.
eduwww.
addgene.
org/CRISPR/reference/#gRNAtools.
flycrispr.
molbio.
wisc.
edu/targetFinderwww.
e-crisp.
org/E-CRISP/designcrispr.
htmlwww.
rgenome.
net/cas-offinderCRISPRtechnologyisrecentandrapidlyevolving.
Onlineresourcesarelikelytochange,asimprovementsandnewtoolsareintroduced.
aUsefulpracticaladviceandaprotocol(appliedtocelllines)canalsobefoundin[129].
GillesandAverofEvoDevo2014,5:43Page8of13http://www.
evodevojournal.
com/content/5/1/43genetics',examiningphenotypesdirectlyintheinjectedembryos(forexample,[48,98,122,130,131]).
Phenotypicanalysiswithoutcrossescouldbeusedasa'quickanddirty'approachinspeciesthathavelonggenerationtimes,orforpreliminaryscreensonalargenumberofcandidategenes,similartoRNAi.
Theobviousdrawbackofthistypeofanalysisisthegeneticmosaicismoftheorganism,whichisdifficulttocontrolandwillleadtopartialandvariablephenotypes.
However,mosaicismcouldalsobeanadvantageincontextswheregeneticmanipulationwithinspecificcelllineagesorinrandomcellclonesisdesirabletoover-comelethality,ortoassesscellautonomousversusnon-autonomouseffectsofgenefunction.
Particularlysowhentheextentofmosaicismcanbemonitoredormanipulated(see[48]).
Tissue-andstage-specificknock-outsmaybeachievedbymanipulatingtheexpressionofCas9[48].
Knock-inapproachesprovideanevenwiderrangeofop-portunities,includingprecisemodificationofgenesintheirnativegenomiccontext,andgeneratingvisiblereportersforregulatoryandphysiologicalevents,anddriversfortransgeneexpression.
Themajorchallengetoovercomehereisthat,foranygivenguideRNAorTALEN,thefrequencyofmutagenicNHEJrepairwillbemuchhigher(byanorderofmagnitude)thanthefrequencyofHDR-orNHEJ-drivenknock-in(seeFigure1).
Ahighknock-outfrequencyinthesomaticcellsofinjectedanimalscanleadtolethalitythatpreventsknock-instoberecoveredinthenextgeneration.
Thisproblemcouldbeovercomeinanumberofways:byusinggermline-specificcis-regulatoryelementstorestricttheactivityofCas9tothegermline[48,123];bytargetingsitesthatareunlikelytobelethalifmutated,forexample,targetingknock-instointronicsequences,withappropriatesplicesignalstogeneratefunctionalgenefusions;byfindingwaystoimprovetheefficiencyofHDRrelativetoNHEJ,suchasbyknockingdowntheactivityofDNAligase4orotherfactorsthatareessentialforNHEJ[49,50,132];orbydevelopingstrategiesthatexploitNHEJ-mediatedgeneknock-instoachievehigherefficiencies[42-45](seeFigure1).
Beyondknock-outandknock-instrategies,theabilitytodirectdouble-strandbreakstospecificsitesinthegenomeraisestheprospectofchromosomeengineering.
Generatingchromosomaldeletionsandinversionsispresentlynotoneoftheusualtoolsemployedinevo-devo,butoneonlyneedstoconsidertheenormouscontributionofbalancerchromosomesinDrosophilageneticstoappre-ciateitspotentialvalueinemergingmodelorganisms[133].
Balancersareinvaluabletoolsforselectingandkeepingtrackofchromosomesbearingmutations,es-peciallywhenthesemutationsaredeleteriousandnotassociatedwithadominantmarker(forexample,theknock-outofanessentialgene).
CRISPRandTALENsnowallowustogeneratetargetedchromosomalinver-sions[134-136]associatedwithrecessivelethalmutationsand,thus,tocreatebalancers,inorganismswhereagen-omesequenceandmapareavailable.
Lastbutnotleast,thecustomizablespecificityofCRISPRmaybeharnessedtodirectothermoleculareffectors-besidesnucleases-tospecificsitesinthegenome(reviewedin[137]).
Forexample,catalyticallyinactiveversionsofCas9havebeenusedtointerferewithtranscription[138,139],coupledwithtranscrip-tionalactivators,repressorsorchromatinmodifierstogenerateartificialtranscriptionalregulators[117,139-143],orlinkedwithfluorescentproteinstorevealchromosomedynamics[144].
Thisapproachallowsustomanipulatetheactivityofregulatoryelementsintheirnativecontextwithoutintroducingchangesintheirnucleotidesequence,providingtoolsofunprecedentedprecisioninoureffortstomanipulateandtounderstandgeneregulation.
CRISPRtechnologyisyoung-barelytwoyearsold-andstillrapidlyevolving.
Improvements,newapplica-tionsandadaptationsofthetechniquetonewspecieshavebeenpublishedatoverwhelmingspeedduringthelastyear,andsurelymoreareforthcoming.
Thisisatruerevolutionforcomparativestudies.
Practicalissues,suchasthedeliverymethodandourabilitytoselectandtopropagatemutants,arestilllikelytolimitthefullde-ploymentofCRISPRinmanyspecies.
Notwithstandingtheseissues,targetedmutagenesisandprecisegeneeditingarenowwithinreachinaverywiderangeoforganisms.
EndnotesaItisinterestingtonotethattheadaptivediversifica-tion,specificityandefficiencyofimmunitymechanismsprovidethebasisforsomeofthemostpowerfultoolsinmolecularbiology:restrictionenzymes,antibodies,RNAiandCRISPR.
bSomewhatshortersequencesmayalsobeused(seeOff-targeteffects).
AbbreviationsCas:CRISPR-associatedprotein;CRISPR:Clusteredregularlyinterspacedshortpalindromicrepeats;crRNA:CRISPR-derivedRNA;HDR:Homology-directedrepair;NHEJ:Non-homologousendjoining;PAM:Protospaceradjacentmotif;RNAi:RNAInterference;TALEN:TALeffectornuclease;tracRNA:Trans-actingantisenseRNA;ZFN:Zinc-fingernuclease.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsAFGandMAwrotethemanuscript.
Bothauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsWethankThomasAuer,FilippodelBene,FrédéricFlamant,MartinKlingler,NikosKonstantinides,ZachariasKontarakis,TassosPavlopoulos,MiltosTsiantisandmembersofourlabforreadingandcommentingonthemanuscript.
OurworkissupportedbygrantANR-12-CHEX-0001-01oftheAgenceGillesandAverofEvoDevo2014,5:43Page9of13http://www.
evodevojournal.
com/content/5/1/43NationaledelaRecherche,bytheMarieCurieITNnetwork'NEPTUNE'andbyafellowshipfromtheUniversitéClaudeBernardLyon1.
Authordetails1InstitutdeGénomiqueFonctionnelledeLyon(IGFL),coleNormaleSupérieuredeLyon,46Alléed'Italie,Lyon69364,France.
2BMICgraduateprogrammeandUniversitéClaudeBernard-Lyon1,Lyon,France.
3CentreNationaldelaRechercheScientifique(CNRS),Lyon,France.
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