===LentiCRISPRv2构建说明书

  Page1of2rev20140722LentiCRISPRv2andlentiGuide-Puro:lentiviralCRISPR/Cas9andsingleguideRNACRISPR(ClusteredRegularlyInterspacedShortPalindromicRepeats)isamicrobialnucleasesysteminvolvedindefenseagainstinvadingphagesandplasmids.CRISPRlociinmicrobialhostscontainacombinationofCRISPR-associated(Cas)genesaswellasnon-codingRNAelementscapableofprogrammingthespecificityoftheCRISPR-mediatednucleicacidcleavage.LentiviralCRISPR/Cascaninfectabroadvarietyofmammaliancellsbyco-expressingamammaliancodon-optimizedCas9nucleasealongwithasingleguideRNA(sgRNA)tofacilitategenomeediting(Shalem*,Sanjana*,etal.,Science2014).Protocolsforcloningintothelentiviraltransferplasmidandgeneralconsiderationsforproducinglentivirusaredescribedbelow.Separateprotocolsareavailableforamplifyingthegenome-scaleCRISPRknock-out(GeCKO)libraries.ThisprotocolisforcreatingindividuallentiviralCRISPRplasmidstargetingasinglegenomiclocus.lentiCRISPRv2(onevectorsystem):Thisplasmidcontainstwoexpressioncassettes,hSpCas9andthechimericguideRNA.ThevectorcanbedigestedusingBsmBI,andapairofannealedoligoscanbeclonedintothesingleguideRNAscaffold.Theoligosaredesignedbasedonthetargetsitesequence(20bp)andneedstobeflankedonthe3'endbya3bpNGGPAMsequence,asshownonthenextpage.lentiGuide-Puro(twovectorsystem):ThisplasmidexpressedonlythechimericguideRNA.ItdoesnotcontainCas9.PleaseuselentiCas9-Blast(aseparatelentiviralconstructthatdelivershSpCas9andblasticidinresistance)tofirstintegrateCas9intoyourcellline.ThelentiGuide-PurovectorcanbedigestedusingBsmBI,andapairofannealedoligoscanbeclonedintothesingleguideRNAscaffold.Theoligosaredesignedbasedonthetargetsitesequence(20bp)andneedstobeflankedonthe3'endbya3bpNGGPAMsequence,asshownonthenextpage.Whichvectortouse:lentiCRISPRv2isidenticaltotheoriginallentiCRISPRv1butproducesnearly10Xhighertitervirus.lentiGuide-Puroproduces100XhighertitervirusoverlentiCRISPRv1andshouldbeusedincelllineswhereCas9hasalreadybeenintegratedin(e.g.usingtheseparatelentiCas9-Blastlentivirus).ForapplicationswhereCas9cannotfirstbeintroduced(e.g.primarycells),lentiCRISPRv2isrecommended.Aftertransduction,usepuromycintoselectforcellswithlentiCRISPRv2orlentiGuide-Puro.Lentiviralproduction:Beforestartinganylentiviralwork,pleaseensurecompliancewithyourEnvironmentalHealthandSafetyofficeandgovernment/organization/university.Briefly,tomakelentivirus,atransferplasmid(e.g.lentiCRISPRv2orlentiGuide-Puro)mustbeco-transfectedintoHEK293(F)TcellswiththepackagingplasmidspVSVg(AddGene8454)andpsPAX2(AddGene12260).Asapositivecontrolforviralproduction,weoftenuseaCMV-EGFPlentiviraltransferplasmid(eg.AddGene19319).Targetdesignnotesandonlineresources:ForapplicationofCas9forsite-specificgenomeeditingineukaryoticcellsandorganisms,wehavecomputationallyidentifiedsuitabletargetsitesfortheS.pyogenesCas9andcalculatedmostlikelyoff-targetswithinthegenome.Pleasevisit::Pleasereferencethefollowingpublicationsfortheuseofthismaterial.Improvedlentiviralvectorsandgenome-widelibrariesforCRISPRscreening.SanjanaNE*,ShalemO*,ZhangF.NatureMethods(2014).Genome-scaleCRISPR-Cas9knockoutscreeninginhumancells.ShalemO*,SanjanaNE*,HartenianE,ShiX,ScottDA,MikkelsenT,HecklD,EbertBL,RootDE,DoenchJG,ZhangF(2014).Science,343,83-7.DOI:10.1126/science.1247005  Page2of2rev20140722TargetGuideSequenceCloningProtocolInordertoclonethetargetsequenceintothelentiCRISPRv2orlentiGuide-Purobackbone,synthesizetwooligosofthefollowingform.AllplasmidshavethesameoverhangsafterBsmBIdigestionandthesameoligoscanbeusedforcloningintolentiCRISPRv2,lentiGuide-PuroorlentiCRISPRv1.Exampleoligodesign:NotethattheNGGPAMisnotincludedinthedesignedoligos.Oligonucleotideorderingtips:Standardde-saltedoligos(usuallythemostinexpensivesynthesis)aresufficientforcloning.Ifnotalreadyresuspended,diluteeacholigoto100µMinsterilewaterorTE.*****Lentiviralvectordigestion,oligoannealingandcloningintodigestedvector:1.Digestanddephosphorylate5ugofthelentiviralCRISPRplasmidwithBsmBIfor30minat37C:5uglentiCRISPRv2orlentiGuide-Puro3ulFastDigestBsmBI(Fermentas)3ulFastAP(Fermentas)6ul10XFastDigestBuffer0.6ul100mMDTT(freshlyprepared)XulddH2O60ultotal2.GelpurifydigestedplasmidusingQIAquickGelExtractionKitandeluteinEB.IfBsmBIdigested,a~2kbfillerpieceshouldbepresentonthegel.Onlygelpurifythelargerband.Leavethe2kbband.3.Phosphorylateandannealeachpairofoligos:1ulOligo1(100µM)1ulOligo2(100µM)1ul10XT4LigationBuffer(NEB)6.5ulddH2O0.5ulT4PNK(NEBM0201S)10ultotalPleaseusetheT4LigationBuffersincethebuffersuppliedwiththeT4PNKenzymedoesnotincludeATP(orsupplementto1mMATP).Putthephosphorylation/annealingreactioninathermocyclerusingthefollowingparameters:37oC30min95oC5minandthenrampdownto25oCat5oC/min4.DiluteannealedoligosfromStep3ata1:200dilutionintosterilewaterorEB.5.Setupligationreactionandincubateatroomtemperaturefor10min:XulBsmBIdigestedplasmidfromStep2(50ng)1uldilute