LentiCRISPRv2构建说明书_精品文档资料下载.pdf
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LentiCRISPRv2构建说明书_精品文档资料下载.pdf
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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)andneedstobeflankedonthe3endbya3bpNGGPAMsequence,asshownonthenextpage.lentiGuide-Puro(twovectorsystem):
@#@ThisplasmidexpressedonlythechimericguideRNA.ItdoesnotcontainCas9.PleaseuselentiCas9-Blast(aseparatelentiviralconstructthatdelivershSpCas9andblasticidinresistance)tofirstintegrateCas9intoyourcellline.ThelentiGuide-PurovectorcanbedigestedusingBsmBI,andapairofannealedoligoscanbeclonedintothesingleguideRNAscaffold.Theoligosaredesignedbasedonthetargetsitesequence(20bp)andneedstobeflankedonthe3endbya3bpNGGPAMsequence,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.Pleasevisithttp:
@#@/www.genome-engineering.orgtoaccesstheseCas9targetdesigntools.Completeplasmidsequences,protocols,adiscussionforumandadditionalinformationcanbefoundattheZhangLabGeCKOwebsite:
@#@http:
@#@/www.genome-engineering.org/gecko/.Citation:
@#@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.1247005Page2of2rev20140722TargetGuideSequenceCloningProtocolInordertoclonethetargetsequenceintothelentiCRISPRv2orlentiGuide-Purobackbone,synthesizetwooligosofthefollowingform.AllplasmidshavethesameoverhangsafterBsmBIdigestionandthesameoligoscanbeusedforcloningintolentiCRISPRv2,lentiGuide-PuroorlentiCRISPRv1.Exampleoligodesign:
@#@NotethattheNGGPAMisnotincludedinthedesignedoligos.Oligonucleotideorderingtips:
@#@Standardde-saltedoligos(usuallythemostinexpensivesynthesis)aresufficientforcloning.Ifnotalreadyresuspended,diluteeacholigoto100MinsterilewaterorTE.*Lentiviralvectordigestion,oligoannealingandcloningintodigestedvector:
@#@1.Digestanddephosphorylate5ugofthelentiviralCRISPRplasmidwithBsmBIfor30minat37C:
@#@5uglentiCRISPRv2orlentiGuide-Puro3ulFastDigestBsmBI(Fermentas)3ulFastAP(Fermentas)6ul10XFastDigestBuffer0.6ul100mMDTT(freshlyprepared)XulddH2O60ultotal2.GelpurifydigestedplasmidusingQIAquickGelExtractionKitandeluteinEB.IfBsmBIdigested,a2kbfillerpieceshouldbe
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