文档介绍：该【fluorescent gene tagging of transcriptionally silent genes in hipscs brock roberts资料 】是由【赖大文档】上传分享，文档一共【14】页，该文档可以免费在线阅读，需要了解更多关于【fluorescent gene tagging of transcriptionally silent genes in hipscs brock roberts资料 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..Pleasecitethisarticleinpressas:Robertsetal.,aggingofTranscriptionallySilentGenesinhiPSCs,StemCellReports(2019),https:///.,,1JoyArakaki,,1HaseebMalik,1AngeliqueNelson,1JamieGehring,1CarolineHookway,,1RuianYang,1AmandaHaupt,1TanyaGrancharova,1VeronicaValencia,,1AndrewTucker,,,*1AllenInstituteforCellScience,Seattle,WA98109,USA*Correspondence:******@https:///.(hiPSCs).AmonomericEGFP(mEGFP)fusiontagandaconstitutivelyexpressedmCherry?uorescenceselectioncassetteweredeliveredintandemviahomology-erefunction:TTN,MYL7,MYL2,TNNI1,-crohomology-mediatedend-joiningandnon-homologousend-,andallproperlytaggedclonesexpressedthemEGFPfusionproteinupondifferentiationintocardiomyocytes,,.,2017).bi-nasestotheeditedcellsandleavesresidualsequencesGenomeeditinghasrevolutionizedcellbiologywiththe(.,LoxPsites)atthetargetlocus,(etal.,2014,2018;DoyonTocircumventthislimitation,wedevelopedaCRISPR/etal.,2011;Grassartetal.,2014;al.,2014;OtsukaCas9editingapproachincorporatinga?uorescenttagetal.,2016;Robertsetal.,2017).Editinghumaninducedintosilentlociwithpotentiallynoresidualsequence(Fig-pluripotentstemcells(hiPSCs)providesaparticularlyure1A).First,amonomericEGFP(mEGFP)tagisdeliveredanizationviahomology-directedrepair(HDR)intandemwithaanddynamics,diseasemechanisms,andregenerationinconstitutivelyexpressedCAGGS:mCherryexcisableselec-adiploid,non-transformed,andrelativelystablegenomictioncassette,enablingFACS---richsequencesintheandregenerativemedicine(DrubinandHyman,2017).donortemplatethatpromoteexcisionviathemicrohomol-WedescribedaCRISPR/Cas9-mediatedendogenousgeneogy-mediatedend-joining(MMEJ)-framepeptidelinkerbetweenthan25differenttargetgenesinhiPSCs(Addgene,2018;thecodingsequenceofthetargetgeneandthe?uorescentCoriellMedicalInstituteBiorepository,2018;Hauptetal.,tag,ratherthananundesiredgenomicscaroftenassociated2018;Robertsetal.,2017).,bypassingartifactscloneswereproductsofMMEJ-mediatedexcision,othersassociatedwithoverexpressionsystems(al.,resultedfromnon-homologousend-joining(NHEJ)-medi-2013).Akeyfeatureofthismethodisthedrug-freeenrich-atedexcisionincorporatingthetetrapeptidePro-Gly-Ser-mentofeditedcellsby?uorescence-activatedcellsortingGlyaspartofthelinkersequence(Figure1A,lowerbox).(FACS),whichreliesontheexpressionofthetaggedpro-,targetgenesofinterestthataresilentinNHEJresultinef?-MMEJhasbeenusedtopromotevariousgenomeeringconstitutivelyexpressedselectionmarkerssuchasmanipulations,includinglargechromosomalinsertions,drugresistanceand/or?-deletions,anddisease-relatedchanges(Baeetal.,2014;quentremovaloftheseselectioncassettesfromtheeditedKimetal.,2018;Nakadeetal.,2014;Sakumaetal.,2016;al.,2018).OurstrategyharnessestheMMEJrepairsuchastheCre/Loxsystem(Skarnesetal.,2011;Yaopathwayforthepurposeofendogenoustagging,–14jMay14,-NC-NDlicense(/licenses/by-nc-nd//).:..Pleasecitethisarticleinpressas:Robertsetal.,aggingofTranscriptionallySilentGenesinhiPSCs,StemCellReports(2019),https:///.-MediatedDeliveryandSubsequentCas9/MMEJ-MediatedExcisionofaConstitutivelyExpressedSelectionCassetteAchievesTaggingofSilentGenes(A)??PAM-out.???ectwork?owvariationsdiscussedinResults.(B)FACSsortingofmCherry+cellsafterHDRattheMYL2andMYL7exampleloci(otherlocishowninFigureS1A).PercentagesofmCherry+cellsisolatedbyFACSaftertransfectionwithdonorplasmidsandtheindicatedduplexedcrRNAandwild-typeCas9RNParedisplayedalongsidemocktransfectionswithnon-?ningmCherry+.(C)Grapheddatafrom(B),errorisstandarddeviation(SD)(FigureS1A).(D)Flow-+cellsfrom(B)wereexpandedandtransfectedwitheithermockorTia1L-speci?+cellpopulations(option1)weretransfectedforexcisionwhileMYL7mCherry+cellswerederivedfromvalidated,unexcisedclones(option2).RNPswereformulatedwith(legendcontinuedonnextpage)–14jMay14,2019:..Pleasecitethisarticleinpressas:Robertsetal.,aggingofTranscriptionallySilentGenesinhiPSCs,StemCellReports(2019),https:///.(RNP)-mediatedelec-havealsousedimprovedgene-RISPRRNAs(crRNAs)excisionef?ciencies(>50%inoptimizedexperiments,inparalleltransfections(Figure1A,‘‘Transfection1’’)(Linwithoutnegativeselection)binases,result-etal.,2014;Robertsetal.,2017;Hauptetal.,2018).-cytometrywasusedtoevaluatethefrequencyofmCherry+stratethismethodbyintroducinganin-framemEGFPtagtocellsindicativeofHDR-mediatedediting(Figures1B,1C,thecodingsequenceof?vetranscriptionallysilentgenesandS1A–S1D).mCherry+-paredwithmockobservedexpressionofthesetaggedproteinsduringcardio-transfections,+cellsmyocytedifferentiationandpreciselocalizationinallcaseswereobservedatafrequency(%–5%)consistentericstructuresinlivecells:theZdiscwithourpreviousstudytagginggenesexpressedinplurip-(ACTN2,a-actinin2),thin?lament(TNNI1,troponinI1),otentcells(Robertsetal.,2017).Therelativelylowratesofatrial(MYL7,myosinlightchain7),ebysorting(MYL2,myosinlightchain2)thick?laments,andtheMasuf?cientnumberofeditedcells(>500)forexpansionandline(TTN,ere-spanningproteintitin,-linelabeling).LiveimagingoftheseimproveHDRrateswithnon-selectivechemicalsthatcellsbypassestheneedtosacri?cesamplesfor?xation,mayadverselyaffectthehealthandpluripotencyofstemcreatingcelllinesthatcanbeusedtostudydynamicbiolog--,wereplacedthePGKpro-Thesemethodsforcreatingisogenic,taggedhiPSCsmaymoterusedininitialexperiments(TNNI1andACTN2)facilitatethestudyofdifferentiation-speci?cproteinsinwithCAGGSformorerobustandstableexpressionoftheresponsetoenvironmental,drug,icperturba-mCherrycassette(FiguresS1A–S1D).?owsforclonegenerationbeforeandafterexci-Thebroadlyapplicabletaggingstrategydescribedheresion(options1and2inFigure1A).Inoption1,mCherry+willthuspotentiallyenablevariousimage-based,biochem-cellsweresorted,expanded,,andfunctionalinvestigationsintodevelopment,dis-TheexcisedmCherrycellswereresorted,expanded,andease,(Figures2A–2Cand2G).Inanalternativework-RESULTS?ow(Figure1A,option2)intendedtoidentifypreciselyeditedclones(.,lackingdonorbackboneormutationsattheuntaggedallele)priortotheexcisionstep,mCherry+HDRTargetingwiththeExcisableCassetteDonorplasmidscontainedtheintendedmEGFPtagcellswereenrichedviaFACSandplatedforclonalisolation?ankedby1-kblocus-speci?chomologyregionsandandscreening(MYL7andACTN2,Figures2D–2G).Onlyincludedthefollowingfeatures:(1)aconstitutivelyex--kbCAGGS:mCherryselectioncassettetofacili-(mCherry+)cellsafteritsdirectedincorporationalongwithmEGFPviaHDR;(2)invertedExcisionofthemCherrySelectionCassettewith‘‘PAM-out’’Tia1LprotospacersabsentfromthehumanCRISPR/Cas9andNHEJ-andMMEJ-MediatedRepairgenomeand?ankingtheselectioncassetteforexcisionaf-esterinitialFACSenrichmentsuchthatNHEJwouldproducePopulationsorclonesofsortedmCherry+cells(options1anin-framemEGFPfusionwithaPro-Gly-Ser-Glylinkerand2)plexesspeci?cto(Lackneretal.,2015);(3)microhomology-containingse--esanddeletetheresidualtioncassettewouldcreateanin-framefusionofthemEGFPsequenceremainingafterCas9cleavage(Figure1A).tagateachendogenouslocus(Figure1A)andresultinduplexedcrRNA:tracrRNAandwild-typeCas9(??Standard??)orwithSynthegosgRNAsandTrueCutCas9(??Optimized??),-.(E)Grapheddatafrom(D).–14jMay14,20193:..Pleasecitethisarticleinpressas:Robertsetal.,aggingofTranscriptionallySilentGenesinhiPSCs,StemCellReports(2019),https:///.(A)SchematicillustratingPCRassaysidentifyingpreciselytagged,excisedclones(Figure1A,??Option1??).ddPCR(left,??Step1??)wasusedtoidentifycloneswithmEGFPinsertion(normalizedgenomicmEGFPcopynumber1)andnostableintegrationoftheplasmidortheexcisedmCherrycassette(opynumber<)(seeExperimentalProcedures).TiledjunctionalPCR(??Step2,??middle)screeningofddPCRvalidatedclonesdepictsampli?cationfromfrequentprecise(green)andimprecise(pink)es.(B)opynumbersofthemEGFP,mCherry,andAmpDNAsequencesweredeterminedbyddPCRinexcisedclones,asexplainedin(A).(C)ordingtoddPCR(green)andjunctionalPCR(blue)assaysareshown,amongclonestested.(legendcontinuedonnextpage)–14jMay14,2019:..Pleasecitethisarticleinpressas:Robertsetal.,aggingofTranscriptionallySilentGenesinhiPSCs,StemCellReports(2019),https:///.-appearedwithin3daysoftransfection,andwereuptoquencywithcorrectPCRjunctions(Figures2Band2C).9-fold(MYL7)morefrequentinTia1Ltransfectedpopula-WeadditionallyperformedSangersequencingoftheparedwithmockcontrolsusingthestandardwild-typealleleandrejectedcloneswithmutationsplex(Figures1D,1E,andS1E–S1I).Recovering(Figure2G).cellsafterexcisionofthePGK:mCherryselectioncassetteToevaluateexcisionprecision,weSangersequencedtheplicatedbydecreasedmCherryexpressionover50junctioncontainingtheexcisionsiteinallexcisedclonestimeinmock-excisedcells,motivatingfutureexperimentswithexpected50P