The R229Q mutation of Rag2 does not characterize severe ...

RAG1 or RAG2 mutations are associated with defects in V(D)J ... used for generating knockout or knock-in mice that mimic human diseases. Skiptomaincontent Thankyouforvisitingnature.com.YouareusingabrowserversionwithlimitedsupportforCSS.Toobtain thebestexperience,werecommendyouuseamoreuptodatebrowser(orturnoffcompatibilitymodein InternetExplorer).Inthemeantime,toensurecontinuedsupport,wearedisplayingthesitewithoutstyles andJavaScript. Advertisement nature scientificreports articles article TheR229QmutationofRag2doesnotcharacterizesevereimmunodeficiencyinmice DownloadPDF Subjects SeverecombinedimmunodeficiencyTargetedgenerepair AbstractRAG1orRAG2mutationsareassociatedwithdefectsinV(D)Jrecombinationactivity,causingsevereimmunodeficiencywithawidespectrumofclinicalphenotypes.AR229QmutationofRAG2wasidentifiedinpatientswithseverecombinedimmunodeficiency(SCID)orOmennsyndrome(OS).AlthoughsomefactorsdeterminingtheclinicalfeaturesbetweenSCIDandOSwerenotclear,themolecularmechanismofOSwasstudiedinamousemodelinwhichanEGFPtagisfusedtoRag2withtheR229Qmutation.Todesignthehumandiseasemodelmimickingsevereimmunodeficiency,wegeneratedRag2-R229Qknock-inmicewithoutanepitopetag.MutantmiceshowedimpairedTandBcelldifferentiationwithreducedV(D)Jrecombinationactivity;however,theextenttowhichtheR229Qmutationaffectssevereimmunodeficiencywasnotsevere.WhileRag2-R229QmutationundersomeconditionsmaycausesevereimmunologicalandclinicalphenotypessimilartohumanSCIDorOS,R229Qmutationpersedidnotcausesevereimmunodeficiencyinmice,suggestingthatadditionalfactorsotherthanR229Qmutationarerequiredtoinducesevereimmunodeficiency.Thus,ourreportimpliesthattheeffectsofgeneticbackgroundand/orataggedproteinsequencemayalterthemouseimmunesystem,revealingthemechanismofphenotypicheterogeneityarisingfromanidenticalmutation. DownloadPDF IntroductionTheadaptiveimmuneresponsetoaparticularpathogenreliesonBandTlymphocytes,whichpossessgeneticallyrearrangedandhighlydiverseantigenreceptors1.DuringtheearlystageofBandTcelldevelopment,theygeneratearepertoireofimmunoglobulinsandTcellreceptorsbyrecombiningvariable(V),diversity(D),andjoining(J)genesegmentsofantigenreceptorloci2.EachoftheV,D,andJgenesegmentsisflankedbyrecombinationsignalsequences(RSSs),composedofconservedheptamerandnonamerelementswhichareseparatedbyaspacerofeither12or23base-pairslong2.Thelymphocyte-specificrecombinationactivatinggene1(RAG1)and2(RAG2)initiatetheprocessofV(D)Jrecombinationbyintroducingsite-specificDNAcleavageatthejunctionbetweentheRSSsandtheadjacentcodingsegment3.Thus,theRAGgenesplayanessentialroleintherearrangementofthegenesencodingantigen-specificreceptorsofBandTlymphocytes,therebyfacilitatingthediversityofantigenrecognitionpresentinthematurelymphocytepopulation4.RAGdeficiencyinhumansleadstoseverecombinedimmunodeficiency(SCID),associatedwithacompleteabsenceofmatureBandTlymphocytes5.However,patientswithhypomorphicmutationsinRAG1orRAG2thatdisplaypartialV(D)Jrecombinationactivitycangiverisetoawidespectrumofclinicalandimmunologicalphenotypes,rangingfromOmennsyndrome(OS)toatypicalSCID6.IntypicalOSpatients,circulatingBcellsaremostlyabsent,whereasthelevelsofTcellsarenormalorelevatedwitharestrictedT-cellreceptor(TCR)repertoire7.PatientsaredefinedtopresentatypicalSCIDwhencorrelatedconditionsdonotfullysatisfythecriteriaforOS8.Whileapproximately15%oftheSCID-sufferinginfantsintheUnitedStateshaveRAG1mutations9,theunderlyingmolecularmechanismsofsuchphenotypicheterogeneityremainunclear.Recently,crystalstructureanalysisofRAGproteincomplexrevealedtheroleofconservedresiduesandfrequentlyoccurringmutationsinpatients10,11.BasedonthestructureoftheRAG1-RAG2proteincomplex,missensemutationsleadingtoSCIDorOScanbecategorizedintofourgroups:(1)mutationsdestabilizingthetertiarystructureofRAG1-RAG2;(2)mutationsaffectingpolarresiduesinvolvedinDNAbinding;(3)mutationssurroundingtheactivesites;and(4)mutationslocatedattheinterfaceofRAG1andRAG210.Amongthese,R229ofRAG2thatformssaltbridgeswithD546ofRAG110isregardedtobecriticalforthedevelopmentofSCIDorOSinpatients5,8,12.Inaddition,homozygousRag2-R229Q/Enhancedgreenfluorescentprotein(EGFP)mutantmice(hereafterRag2KI/EGFP),developedbyMarrellaandcolleagues13,presentedclinicalandimmunologicalphenotypesremarkablysimilartohumanOSwithseverealopecia,erythroderma,infiltrationbyTlymphocytesandeosinophilsintotheskinandgut,andcompleteabsenceofBcells.However,thismousemodelexpressedthemutantprotein,inwhichtheendogenousRag2genewastargetedbyaconstructcontainingRag2-R229QwithanEGFPtagattheNterminus.Furthermore,theycreatedthemutantmicebygenetargetingmethodusingmouse129/Svembryo-derivedstem(ES)cellsandperformedanalysisonamixedC57BL/6X129/Svgeneticbackground13.AlthoughtheRag2KI/EGFPmiceusedasmurineOSmodelhelpedustounderstandthedetailedpathogenesisofOSandautoimmunity,whethertheR229QmutationitselfissufficienttocausefulldevelopmentofOSandseverelyaffectimmunologicaldisorderisstillnotclear.Animalmodelshavebeenprovidingvaluablecluestotheaetiologyandthemolecularpathogenesisofhumangeneticdiseasescausedbyseveraltypesofmutations.Recently,aclusteredregularlyinterspacedshortpalindromicrepeat(CRISPR)/Cas9,identifiedinbacteriaandarchaeaastheheritableandadaptiveimmunesystem,hasbecomeapowerfultoolforgenomeeditingineukaryoticcellsduetoitsaccuracy,simplicity,andhighefficiency14.TheCRISPR/Cas9systemiscomposedofCas9endonucleaseandasyntheticsingleguideRNA(sgRNA)thatcontainsatargetingsequence(crRNA)andaCas9nuclease-recruitingsequence[transactivatingcrRNA(tracrRNA)]14.ThesgRNAincludes20nucleotidesthatarecomplementarytoatargetsequenceupstreamofaPAMsequence(NGG)anddirectstheCas9endonucleasetoaspecificlocationinthegenome.TheCas9endonuclease,alongwithansgRNA,introducessequencespecificDNAdouble-strandbreakswhichresultsinnon-homologousendjoining(NHEJ)-mediatedinsertionsanddeletionsorhomology-directedrepair(HDR)-mediatedrepairinthepresenceofdonortemplates.Inmice,CRISPR/Cas9hasbeensuccessfullyusedforgeneratingknockoutorknock-inmicethatmimichumandiseases15.Inthisstudy,wegeneratedRag2-R229Qknock-inmicebyCRISPR/Cas9-mediatedgeneeditinginC57BL/6zygotes.SinceRag2KI/EGFPmousemodelhasbeenusedtostudypathogenesisofOS13andtheR229QresidueofRAG2hasbeenconsideredimportantforlymphocytedevelopmentinpatients5,8,12,ourfirstintentionwastosetthemupasapotentialmodelforgenetherapyofSCIDandOS.However,ourRag2-R229QmutantmousemodelexhibitedamilderimmunologicalphenotypeinlymphocytedevelopmentthaninthepreviousRag2KI/EGFPmousemodel13.Furthermore,wecouldnotobserveclinicalevidenceforOS,possiblyduetotheabsenceofareportergeneand/ortheinfluenceofgeneticbackground.Accordingly,ourresultsclearlydemonstratedthatidenticalmutationinRAG2giverisetodistinctinvivophenotypesdependingonexternalfactors,whiletheR229QmutationofRag2byitselfisnotsufficienttocausethefulldevelopmentofOSandSCIDinmice.ResultsGenerationofRag2-R229QmicebyCRISPR/Cas9-mediatedgeneeditingToestablishamurinemodelofsevereimmunodeficiencywithSCIDorOS,wegeneratedaRag2knock-inmousecarryingtheR229QmutationbyCRISPR/Cas9-mediatedgeneediting.TheR229residueofRAG2,frequentlyidentifiedinpatientswithSCIDorOS,isconsideredtobecriticalforthedevelopmentofSCIDorOS5,8,12.Inaccordancewiththis,apreviouswork13hasshownthatRag2KI/EGFPmicedisplayedimmunologicalandclinicalphenotypessimilartothoseofhumanOS;however,theygeneratedmutantmiceinwhichanEGFPtagisfusedtoendogenousRag2andwerebredonamixedC57BL6X129/Svgeneticbackground.Toremovetheeffectsofgeneticbackgroundandanartificialtag,multiplesgRNAsandasingle-strandedoligodeoxyribonucleotide(ssODN)weredesignedfortargetingtheexon3ofRag2tointroducetheR229QmutationonthepureC57BL/6geneticbackground(Fig. 1A).WeconstructedthessODNwithsilentmutationstopreventre-cleavingbyCRISPR/Cas9andtoaidPolymerasechainreaction(PCR)-basedgenotypemethods.Afterco-injectionofCas9mRNA,sgRNAs,andssODNintopureC57BL/6zygotes,wescreenedthefoundermicebyPCRamplificationontheRag2locus.Among27newborns,weobtainedonetargetedknock-inmouse(SupplementaryTable S1).Totestgermlinetransmissionandreducepotentialriskofoff-targeteffects,themutantmousewascontinuallybredwithC57BL/6wild-typemiceandconfirmedthepresenceoftheRag2-R229Qmutationintheirprogenythroughgenomicsequencing(Fig. 1B).Theheterozygousmutantmiceweresubsequentlyintercrossedtogeneratehomozygousmutantmice(Fig. 1C).ThehomozygousRag2-R229Qknock-in(hereafterRag2KI)micewerefertileandsurvivedintoadulthoodwithnoobviousdevelopmentdefects,comparedtowild-typestrains(hereafterRag2WT;datanotshown).ItisworthwhiletonotethatunlikeRag2KI/EGFPmice,ourRag2KImicedidnotshowanypathologicalphenotypesofhumanOSsuchasseverealopeciaandskinerythroderma(SupplementaryFig. S1).Figure1IntroductionoftheR229QmutationinRag2locusbyCRISPR/Cas9-mediatedgeneediting.(A)AschematicrepresentationofsgRNAtargetingsitesinRag2andsequenceofssODN.ThesgRNAtargetsandPAMsequencesareshowninblueandred,respectively.The173-bpssODNdonortemplateispresentedunderthegenomicDNAwithaR229Qmutantsequenceingreen.Thesilentmutationsusedtopreventre-cuttingbyCas9areunderlined,whereastheboldandunderlinedsequencesarethessODNdonortemplatesequencesthatweresuccessfullyintroducedintoRag2locus.(B)PCRanalysisofgenomicDNAfromwild-typeandRag2-R229Qknock-inmice.SpecificbandswereobservedaftergenotypingattheexpectedDNAsize:Rag2wild-type(WT) = 300 bp;Rag2knock-in(KI) = 188 bp.(C)ComparisonofgenomicDNAsequencesbetweenRag2wild-typeandRag2R229Qmutation.FullsizeimageAmildimpairmentofearlyTcelldevelopmentinRag2-R229Qknock-inmiceToinvestigatetheeffectsoftheRag2-R229QmutationonTcelldevelopment,weperformedfluorescence-activatedcellsorting(FACS)analysisforthymocytesinRag2KImice.DuringTcelldevelopment,immatureTcellprogenitorsinthethymusprogressfromtheCD4−CD8−double-negative(DN)totheCD4+CD8+double-positive(DP)stage.AccordingtopreviousRag2KI/EGFPmousemodel,mostthymocyteswereatDNstages(3%inRag2WTversus89%inRag2KI/EGFP)13.Incontrast,onlyminorpopulationofthymocytesfromRag2KImicewasobservedatDNstage(3.8%inRag2WTversus15.5%inRag2KI,Fig. 2A).Nevertheless,wefoundthatRag2KImiceshowedamarkedincreaseinthenumberandpercentageofDNpopulation,suggestingthatthymocytedevelopmentwaspartiallyarrestedattheDNstage(Fig. 2AandSupplementaryFig. S2).TheDNpopulationisdividedaccordingtotheexpressionofCD44andCD25,intofoursubsets:CD44+CD25−(DN1),CD44+CD25+(DN2),CD44−CD25+(DN3),andCD44−CD25−(DN4)16.TCRβrearrangementisinitiatedbyDβ-JβrecombinationduringtransitionfromtheDN2toDN3stageandcompletedbyVβ-DJβjoiningattheDN3stage17.FurtheranalysisrevealedthattheDN3cellnumbersfromRag2KImicewerehighlyaccumulated,whereastheDN4cellnumberswerereduced,indicatingthearrestofthymocytedevelopmentattheDN3stage(Fig. 2BandSupplementaryFig. S2).WhilemicelackingeitherRAG1orRAG2displayedacompleteblockinthymocytedevelopmentattheDN3stage18,19,ourdatasupportthattheR229residueofRag2affectsrearrangementofTCRβlocus.Figure2ImpairedTcelldevelopmentinRag2-R229QKImice.Flowcytometricanalyseswereperformedonwild-type(hereafterRag2WT)andhomozygousRag2-R229Qknock-in(hereafterRag2KI)mice.(A)ThymocyteswerestainedwithCD8andCD4antibodies.Percentagesofeachquadrantaresummarizedinthebargraph(n = 8).DN,Doublenegative;DP,Double-positive.(B)CD4−CD8−(DN)cellsweregatedandstainedwithCD25andCD44antibodies.Percentagesofeachquadrantweresummarizedinthebargraph.DN1,CD44+CD25−;DN2,CD44+CD25+;DN3,CD44−CD25+;DN4,CD44−CD25−.(C)CD4+CD8+(DP)cellsweregatedandstainedwithTCRβandCD69antibodies.Numbersintheplotsindicatethepercentagesofcellsineachquadrant.Percentagesofeachquadrantaresummarizedinthebargraph.(D)TCRβexpressionsontheindicatedthymocytesubsetswereanalysedbyflowcytometry.Gray-tintedhistograms,Rag2WT;blackhistograms,Rag2KI.Bargraphsshowmean ± standarderrorofthemean(SEM).n = 8pergroup.***p