LentiCRISPRv2构建说明书.pdf

LentiCRISPRv2构建说明书.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,a2kbfillerpieceshouldbepresentonthegel.Onlygelpurifythelargerband.Leavethe2kbband.3.Phosphorylateandannealeachpairofoligos:

1ulOligo1（100M）1ulOligo2（100M）1ul10XT4LigationBuffer（NEB）6.5ulddH2O0.5ulT4PNK（NEBM0201S）10ultotalPleaseusetheT4LigationBuffersincethebuffersuppliedwiththeT4PNKenzymedoesnotincludeATP（orsupplementto1mMATP）.Putthephosphorylation/annealingreactioninathermocyclerusingthefollowingparameters:

37oC30min95oC5minandthenrampdownto25oCat5oC/min4.DiluteannealedoligosfromStep3ata1:

200dilutionintosterilewaterorEB.5.Setupligationreactionandincubateatroomtemperaturefor10min:

XulBsmBIdigestedplasmidfromStep2（50ng）1uldilutedoligoduplexfromStep45ul2XQuickLigaseBuffer（NEB）XulddH2O10ulsubtotal1ulQuickLigase（NEBM2200S）11ultotalAlsoperformanegativecontrolligation（vector-onlywithwaterinplaceofoligos）andtransformation.6.TransformationintoStbl3bacteria.LentiviraltransferplasmidscontainLong-TerminalRepeats（LTRs）andmustbetransformedintorecombination-deficientbacteria.WeusehomemadeStbl3（propagatedfromInvitrogenC7373-03）andgetexcellentplasmidyields.AlthoughotherRecA-strainsmaywork,wehavefoundthemostconsistenttransformationsandyieldsusingStbl3.