JosepMonserrat andPaolaScaf CancerEpigeneticsLaboratoryTheFrancisCrickInstitute1MidlandRoadLondonNW11ATUKUCLCancerInstituteUniversityCollegeLondonLondonWC1E6DDUKARTICLEHISTORYReceived1Septemb ID: 936425
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RESEARCHPAPER ,JosepMonserrat andPaolaScaf CancerEpigeneticsLaboratory,TheFrancisCrickInstitute,1MidlandRoad,LondonNW11AT,UK;UCLCancerInstitute,UniversityCollegeLondon,LondonWC1E6DD,UKARTICLEHISTORYReceived1September2017Revised11October2017Accepted13October2017ABSTRACTTheCRISPR-Cas9systemhasrevolutionizedgenomeengineering,allowingprecisemodicationofDNAinvariousorganisms.ThemostpopularmethodforconductingCRISPR-basedfunctionalscreensinvolvestheuseofpooledlentivirallibrariesinselectionscreenscoupledwithnext-generationsequencing.Screensemployinggenome-scalepooledsmallguideRNA(sgRNA)librariesaredemanding,particularlywhencomplexassaysareused.Furthermore,pooledlibrariesarenotsuitableformicroscopy-basedhigh-contentscreensorforsystematicinterrogationofproteinfunction.ToovercometheselimitationsandexploitCRISPR-basedtechnologiestocomprehensivelyinvestigateepigeneticmechanisms,wehavegeneratedafocusedsgRNAlibrarytargeting450epigeneticregulatorswithmultiplesgRNAsinhumancells.Thelentivirallibraryisavailablebothinanarrayedandpooledformatandallowstemporally-controlledinductionofgeneknock-out.CharacterizationofthelibraryshowedhigheditingactivityofmostsgRNAsandefcientknock-outattheproteinlevelinpolyclonalpopulations.ThesgRNAlibrarycanbeusedforbothselectionandhigh-contentscreens,aswellasfortargetedinvestigationofselectedproteinswithoutrequiringisolationofknock-outclones.Usingavarietyoffunctionalassaysweshowthatthelibraryissuitableforboth CONTACTPaolaScaf paola.scafdi@crick.ac.uk Supplementaldataforthisarticlecanbeaccessedat whole-genomearrayedlibrariesthatallowcomprehensiveinterrogationofgenefunctionhavebeengenerated[],theyrequirelargeamountsofreagents,automatedequipmentanddedicatedfacilities.Moreover,manyscreensdonotrequireexaminationofthewholegenomeandfocusedlibrariestarget-ingclassesofrelatedproteinsmayprovideamoresuitabletooltoeffectivelyaddressspecicquestions.Epigeneticmechanismsplayacriticalroleindevelopment,tissuehomeostasisandcontributetovariousdiseases,includingcancer,syndromesassociatedwithneurologicaldefectsandheartdisease[[26].Owingtotheirinherentreversibility,epigeneticmechanismsrepresentpromisingtherapeutictargetsandidenticationofnovelepigeneticregulatorsinvolvedindis-easemayopennewavenuesfortherapeuticintervention.Here,wereportthegenerationandcharacterizationofansgRNAlibrarytargetingmostknownhumanepigeneticregulatorswithmultipleguides,suitableforbothselectionandhigh-contentscreens,andcompatiblewithavarietyofinvitroinvivoassays.LibrarydesignEpigeneticregulatorstobetargetedwereselectedbasedonGeneOntology,includingproteinsinvolvedinchromosomeorganization,chromatinmodication,andregulationofDNAmethylation.Additionalproteinswereincludedbasedonoverallsimilaritywithknownepigeneticregulatorsorthepresenceofproteindomainspredictedtobindtoormodifychromatin(e.g.,chromodomain,bromodomainorSETdomain).SelectedproteinsinvolvedinDNAdamageresponseandrepairwerealsoincludedinthelistoftargets.Alltogether,450geneswereselectedfortargeting(Fig.1SupplementaryTable1).Foreachgene,wedesigned5differentsgRNAs(sgRNAgroups15)usingtheCRISPRMITDesigner(crispr.mit.edu)mit.edu)27].Wherepossible,sgRNAsweredesignedtotargetthetranslatedexoncommontoallRefSeqisoformsofeachgeneFig.1BandSupplementaryFig.1A).Tominimizeoff-targeteffects,wepreferentiallyselectedsgRNAswithscoresofatleast70,avaluethattypicallycharacterizeshighlyspecicsgRNAssgRNAs27](Fig.1C).WhentargetingofasharedexonwithaspecisgRNAwasnotpossible,themost5exonoftheprimaryRefSeqvariantwasused.(SupplementaryFig.1A).Asanaddi-tionalcriterion,wheneverpossible,weensuredthattargetshadatleast3non-overlappingguides.Basedontheabovestrategy,thedesignedsetofguidescomprised98%ofsgRNAswithscore70(Fig.1C),74%ofsgRNAstargetingtheN-terminuspartofthecorrespondingproteins(%ofremainingpeptideFig.1D,blueboxes),and90%ofsgRNAswithoptimalGCcontent(35-70%)[Fig.1E,blueh
istogram).Furthermore,analysisofthedesignedsgRNAsusinganalgorithmthatpre-dictsguideefcacy[]showedthat82%ofthesgRNAshadanAzimuthRS2score0.4(range:0.04-0.81),indicatinghighpredictedactivity(Fig.1F,blueboxes).SimilarresultswereobtainedanalyzingthesgRNAsequencesusingRNAScorer2.0,adifferentpredictivealgorithm[](SupplementaryFig.2A,B).Thus,thedesignedsgRNAgroups15arepredictedtogenerateshorttruncatedproteins,withminimaloff-targeteffectsandhighefcacy.Foreachgeneinthelibrary,2additionalguides(sgRNAgroups67)wereselectedfromthewhole-genomeCRISPRlibraryconstructedbyWangetal.,[]whichcomprisessgRNAsprimarilydesignedbasedonpredictedefcacy.UnlikesgRNAsgroups15,thesgRNAgroups67donottargetpref-erentially5exonsandshowedamoreuniformdistributionalonggenes(Fig.1D,yellowboxes),buthadcomparableGCcontentandhigherAzimuthRS2score(Fig.1E-F).Thecom-binedsgRNAgroupsresultedinalibraryof3150uniquesgRNAsequenceswith7sgRNAspertargetgene(Supplemen-taryTable2).Toachieveefcientin/delgeneration,wechoseaselectablelentiviralvectorfordeliveryofsgRNAsintocells.pLen-ti_BSD_sgRNA,generatedbysubstitutingtheEGFPcodingregionofthepLentiplasmid[]withacDNA(BSD)encodingresistancetotheantibioticblasticidin,allowsefcientexpres-sionofsgRNAsunderahumanU6promoter(Fig.2A).Tobeamenableforlargescalecloning,thevectorofchoiceshouldallowfastandefcientcloningofsgRNAs,andeliminateanybackgroundbyemptyvector.pLenti_BSD_sgRNAcontainsrestrictionsites,whichallowefcientplasmiddiges-tionandinsertligationinasinglereactionbyGoldenGatecloning.Moreover,thevectorcontainsasuicidegene,whichisreplacedbythesgRNAuponcloning(Fig.2A),pre-ventingpropagationoftheemptyvectorandallowinghighlycientselectionofbacteriatransformedbythecorrectplas-midwithoutneedingtoplatethemonsolidmedium.LibraryproductionForeachsgRNA,individualforwardandreverseoligostaggedwithappropriate5overhangscompatiblewiththebasedligationweresynthesizedin96-wellformat(2plates).AnnealedoligoswereclonedintopLenti_BSD_sgRNAbyGoldenGatecloning(seemethods)(Fig.2BandSupple-mentaryFig.1B).bacteriaweresubsequentlytransformedwiththeGoldenGatemixesin96-deep-wellblocksandgrown20hat37Fig.2BandSupplementaryFig.1C).Inordertoequalizebacterialgrowthamongwellsandobtaincon-sistentplasmidyield,cultureswerediluted1:5infreshmediumandgrownforadditional3.5h.Analysisofbacterialgrowthbyspectrophotometricmeasurementsindicateduniformcellgrowthacrosswells(SupplementaryFig.1D,E).AnalysisofselectedplatesbySangersequencingofisolatedplasmidscon-rmedthehighefciencyandaccuracy(99%)ofthecloningprocedure(Fig.2C).Productionoftheentirelibrarywascom-pletedin10days.InadditiontogeneratingindividualsgRNAsinanarrayedformat,wealsoproducedtwotypesofpooledlibraries,onecontainingallsgRNAs,tobeusedinselectionscreens(pooledlibrary),andoneinwhichallsgRNAstargetingthesamegenewerecombinedandmaintainedina96-wellformat(genepools,in450wells),tobeusedinhigh-contentscreensFig.2D).Bothpooledlibrariesweregeneratedbycombiningaliquotsofbacterialcultureaftertheadditional3.5hofgrowth.AllindividualandpooledbacterialstocksweredigitallytaggedwithbarcodesandstoredatFig.2 T.HENSER-BROWNHILLETAL. AssessmentofsgRNArepresentationinthelibraryInordertoassesstherelativerepresentationofsgRNAsinthegeneratedlibrary,weperformedhigh-throughputsequencingofthepooledlibraryandcomparedrelativeabundanceofeachguide.Eighty-sevenpercentofthedesignedsgRNAsweredetected,showinganarrowdistributionofreadcountsFig.2E).TherawalignedreadsforthemajorityofsgRNAsequencespresentinthepoolshowedlessthan2-folddiffer-encefromthemean(Fig.2E).AsingleoutlieroverrepresentedcomparedtotheothersgRNAswasfoundtohavecontami-natedneighboringwellsinoneplate(Fig.2E).Sangersequenc-ingofindividualplasmidsisolatedfromre-grownglycerolstocksallowedustoidentifythecontaminatedwellsandreplacethemwiththecorrectplasmids.UndetectedsgRNAswerere-culturedfrombacterialglycerolstocksinindividualwellsof96
-wellblocks,andequalizedtoanOD600of0.80priortoplasmidextraction.Sangersequenc-ingindicatedthat58%ofthesgRNAsundetectedinthepooledlibraryhadbeensuccessfullyclonedandhadthecorrectsequence,suggestingthattheymayhaveonlybeenunderrepre-sentedinthepooledlibrary.PuriedplasmidDNAfromtheoriginallyunderrepresentedwellswasaddedtothepooledlibrary.Thenallibrarycomprised94%ofthedesignedguidesallowingtargetingofall450targetgenes,with99%ofgenestar-getedbyatleast4sgRNAs(Fig.2F,SupplementaryTable2).AssessmentofsgRNAactivityToassesstheeditingefcacyofthegeneratedlibrary,weini-tiallyselected15sgRNAsandexaminedtheirabilitytoinducein/delsattheirtargetsites.Todoso,wetransducedhumanbroblastsexpressinginducibleCas9withindividualconstructsandassessedin/delformationbyTOPOcloningofPCRedgenomicfragmentsofthetargetedloci.FourteendaysafterCas9induction,12/15targetedsitesshowedin/dels,indicatingoverallhigheditingactivity(Fig.3A).Timecourseexperimentsindicatedthatin/delformationincreasedovertimeafterCas9induction,typicallyreachingaplateauat8days(notshown).TocomprehensivelyassesstheabilityofindividualsgRNAstoinducein/delformationatalltargetgenes,wegenerated3poolsof450sgRNAscontainingonesgRNA/geneeach(pool5,pool6,andpool7,eachonecontainingdistinctsgRNAsforthesamegene)(SupplementaryTable2),andtransducedCas9- 50100150200 0255075100DEF Targets: Histone modifiers Chromatin remodelers Histone variants DNA-methyl ransferasest DNA-methyl binding proteins DNA repair factors450 human epigenetic regulators 7sgRNAs each 3150 arrayed sgRNAs 95.2% YesNo 0.250.500.751.00 Predicted Activity (RS2 Score) 255075100 % Remaining Peptide Histone chaperones p 0.000.02 0255075100 Wang Wang design Wang design Figure1.sgRNAlibrarydesignA.BasicinformationaboutthegenestargetedinthesgRNAlibrary.Thenumberandtheclassesoftargetedgenesareindicated..Rela-tiveabundanceofdesignedsgRNAs(groups15)targetingallRefSeqisoformsofthecorrespondinggenes(blue)..DistributionofMITspecicityscoreacrossthedesignedsgRNAgroups1.Distributionoftargetedsiteswithingenes,comparingnewlydesignedsgRNAs(Henserdesign,blue)andsgRNAselectedfromthelibrarydesignedbyWangetal.[](Wangdesign,yellow).Groups15sgRNAs(Henserdesign)targetthemost5exons,resultingintheformationofshorttruncatedproteins.In/delsinducedbygroups67sgRNAs(Wangdesign)leadtolongertruncatedproteins,possiblyretainingpartialfunctionality.value:2-wayANOVA..DistributionofGCcontentofthetargetsequencesacrosstheindicatedsgRNAgroups..PredictedactivityoftheindicatedsgRNAgroupsbasedontheAzimuthalgorithm.value:2-wayANOVA.EPIGENETICS expressingHepG2cellswiththethreeindividualpools.Fivedaysafterinfection,genomicDNAwasextractedandtargetedsitespulled-downusingtarget-enrichmentprobes(seemeth-ods).Detectionofin/delsbyhigh-throughputsequencingcon-rmedhigheditingactivityformostsgRNAs.Eighty-sevenpercentofsgRNAsinduceddetectablein/delsand98.6%ofthegenesweretargetedbyatleastonesgRNA(Fig.3B,C).sgRNAgroups67showedhigheractivity(95%and90%ofactivesgRNAs,respectively)comparedtosgRNAgroup5(79%)(value0.0001Fisherstest),suggestingthatthealgorithmusedbyWangetal.todesignsgRNAsmayoutperformtheMITCRISPRDesignerwithrespecttoeditingefcacy(Fig.3However,highersgRNAactivitywouldnotnecessarilyresultinhigherKOefciencysincesgRNAgroups67donotpreferen-tiallytarget5exons,unlikesgRNAgroups1Fig.1D),andmayleadtolongtruncatedproteinsretainingpartialfunctionality.Thus,thecombinationofmultiplesgRNAsdesignedwithdifferentalgorithmsmaximizesthechanceofachievingefcientgeneKOwithourlibrary.Comparisonbetweenthe50mostactivesgRNAsineachsgRNAgroupandthoseshowingloworundetectableactivityindicatedthatthenumberofin/delsinducedbyindividualsgRNAsdidnotcorrelatewithabundanceofthesgRNAinthepool,GCcontentofthetargetsequenceorspecicityscore(SupplementaryFig.2C).ToquantifytheeditingactivityofeachsgRNApresentinthelibrary,wecalculatedanobservedactivityscore(OA
S:thenumberofobservedin/delsnormalizedtotheabundanceofeachsgRNAinthelibraryseeMaterialsandmethods)(SupplementaryTable2).TheOASshowedacant,albeitweak,correlationwiththeAzimuthRS2score(Kendallrankcorrelationcoefcient=0.3,0.001),indicat-ingthatthealgorithmisoverallpredictiveofsgRNAactivity, Figure2.sgRNAlibrarygenerationA.SchematicrepresentationofthevectorusedtogeneratethesgRNAlibrary.LTR:LongTerminalRepeat;U6:U6promoter;ccdB:sui-cidegene;CAT/CmR:chloramphenicolresistance;BsmBI:restrictionenzyme.CMV:cytomegaloviruspromoter;BSD:blasticidinresistance.ThelowersideofthegraphicsrepresentsthenalconstructcontainingthesgRNA(redrectangle)..Summaryofthethree-steplarge-scalecloningprotocol..Heatmapshowingthefractionofcorrectconstructsinplate1ofthelibraryasassessedbySangersequencing.OnlyonesgRNA,indicatedinwhite,wasnotsuccessfullycloned..Summaryofthethreedifferentformatsinwhichthelibrarywasgeneratedandstored..RelativeabundanceofindividualsgRNAsasassessedbynext-generationsequencingofthepooledlibrary.EachdotrepresentsanindividualsgRNA.TheredlineindicatesthemedianvalueofsgRNAcounts..NumberofgeneswiththeindicatednumberofdesignedsgRNAsrepre-sentedinthelibrary.Thepercentageofgenestargetedbyatleast4sgRNAsisindicated. T.HENSER-BROWNHILLETAL. althoughitreturnsmanyfalsepositivesandfalsenegativesFig.3D,SupplementaryFig.2C).Similarresultswereobtainedcomparingthein/delscorewiththesgRNAScorer2.0(Supple-mentaryFig.2D).Thus,whileinsilicoanalysiscanhelpidentifyactivesgRNAs,actualeditingefcacyneedstobeempiricallydetermined.HighsgRNAefcacywasconrmedattheproteinlevel.ToperformaninitialquantitativeassessmentofKOkineticsandciencyinpolyclonalpopulations,weusedareportercelllineexpressingTurboRFP.FACSanalysisofreportercellstransducedwithTurboRFP-targetingsgRNAsindicatedrapidandefKOattheproteinlevel,detecting70%RFP-negativecells8daysafterCas9induction(Fig.3E).KOoftheendogenousH1F0usingsgRNAsfromthelibraryshowedsimilarresults(90%ofH1.0-negativecells,asassedbyimmunouorescencemicroscopy)(Fig.3F).Thus,stableexpressionofsgRNAsallowscientgeneKOinpolyclonalpopulations,avoidingtheneedofisolatingindividualclonalpopulationsofeditedcells.Sensitivedetectionofphenotypiceffectsinducedbygeneknock-outinpolyclonalpopulationsCRISPR-mediatedKOoffersmajoradvantagescomparedtoRNAi-basedapproaches,allowingcompleteeliminationofthetargetedprotein.However,theloweditingefciencyachievedwhenusingtransientlyexpressedsgRNAsandtheconsequentneedtoisolatecloneslimitstheuseofCRISPRforhigh-throughputapproaches.Furthermore,theinabilitytotempo-rallycontrolinductionofgeneKOwhenCas9/sgRNAcom-plexesaretransientlyexpressed,hinderssomeapplications,especiallyifessentialgenesaretargeted(e.g.,invivostudies).Usingtheplatformthatwehavegenerated,geneKOcanbeinducedinpolyclonalpopulationsinatime-controlledmanner,allowinglargescalegenerationofcelllinessuitableforavarietyinvitroinvivoassays(Fig.4A).ToconrmthatthefunctionalconsequencesofgeneKOarerobustlydetectedinpolyclonalcellpopulations,werstassessedwhetherinterfer-encewithahistonemodifyingproteincomplexledtochangesinthecorrespondinghistonemark.Todoso,wedisruptedtheMSLcomplex,whichspecicallyacetylateshistoneH4lysineK16[],byknocking-outMSL1.TomaximizetheKOefciencywetransducedcellswiththepoolof-targetingsgRNAsthatwegenerated.UponKO,weobservedasig-cantreductioninthelevelsofH4K16Ac(0.001Studentt-test),conrmingMSL1loss-of-functionandindicat-ingthatthemolecularconsequencesinducedbyMSL1werereadilydetectable(Fig.4Wethenaskedwhetherwecouldobservephysiologicalcon-sequencesinducedbygeneKOusingpolyclonalpopulations.Thegeneencodingthechromatinremodelerisanessentialgene,whoselossleadstoembryoniclethalityandimpairscellproliferation[].Inagreement, Figure3.ValidationofthesgRNAlibraryA.AssessmentofsgRNAin/delactivityoftheindicatedguides.AssessmentofactivitywasbasedonSangersequencingofPCR-edandclonedtargetregions..As
sessmentofsgRNAin/delefcacyofallguidesincludedintheindicatedsgRNAgroups.Assessmentofactivitywasbasedonnextgenerationsequencingofpulled-downtargetregions.TheindicatedpercentageofactivesgRNAiscalculatedbasedonthenumberofsgRNAsdetectedintheinfectedcells..Percentageofgeneseditedbyatleast1ofthe3testedsgRNAs..CorrelationbetweentheobservedandpredictedsgRNAeditingactivity.TauindicatestheKendallrankcorrelationcoefcient(tau-b).FACSanalysisofcellsexpressingTurboRFPinthepresence(right)orabsence(left)ofTurboRFP-targetingsgRNAs.uorescencemicroscopyofuninduced(NT)orinduced(DOX)cellsexpressing-targetingsgRNAs.Cellswereanalyzed14daysafterCas9induction.Scalebar:10EPIGENETICS inhibitedcellgrowthand8daysafterCas9inductionweobserveda7-foldreductioninthenumberofviablecellscom-paredwiththeuninducedcontrol(Fig.4Finally,toassesswhethergeneKOinducedwithourplat-formisasuitableapproachtoperforminvivofunctionalstud-ies,wetestedtheeffectofKOinxenograftassays.TodoHRAS-transformedbroblasts[]expressinginducibleCas9and-targetingsgRNAs,wereinjectedintradermallyintoimmunocompromisedmice.Whentumorsbecamepalpa-ble(3weeksafterinjection),doxycycline(Dox)wasadminis-teredtomicetoinduceCas9expressionandgeneKOinvivoInagreementwithpreviousreports[],lossofINO80signicantlyinhibitedtumorgrowth(Fig.4E,F).Asapositivecontrol,KOalmostcompletelypreventedtumorgrowth,indicat-ingthatcellsescapinggeneKO,whicharepositivelyselectedduringtumorgrowth,areonlyasmallfractionoftheinjectedcellsanddonotsignicantlyreducethedynamicrangeofdetectabledifferences(Fig.4E).WehavegeneratedandcharacterizedafocusedsgRNAlibrarytargetingmosthumanepigeneticregulatorsthatenablescom-prehensiveinvestigationofcellularmechanismsinvolvingchromatinandDNAmethylation.Weshowoverallhighedit-ingactivityofthesgRNAscontainedinthelibraryandprovideascoreofactualactivityfor3guides/genetoeaseselectionofthemosteffectivesgRNAs.Thelibrarycanbeusedforavarietyofapplications,rangingfromloss-of-functionscreenstoinves-tigationofselectedproteinsinvitroinvivo,visualizationofgenomicloci[]andinductionofgenomicrearrangementsatcloci[Thelibraryisavailableinthreeformats:apoolcontainingallsgRNAs,suitableforbothenrichmentanddrop-outselectionscreens;anarrayedformatinwhichmultiplesgRNAstargetingthesamegenearecombined,suitableforprimaryhigh-contentscreens;anarrayedformatcontainingindividualsgRNAs,whichcanbeusedtovalidateprimaryhitsortoinvestigateselectedproteinsinatargetedmanner.Importantly,weshowthatthehigheditingefciencyinpolyclonalpopulationsallowsassessmentoffunctionalconsequencesofgeneKOwithouttheneedofisolatingclones.Thisremovesatime-consumingstepandgeneratesmorereliabledata,avoidingclonaleffectsthatcansignicantlyaffectthebiologicalinterpretationoftheresults,especiallywhenheterogeneouscellpopulationssuchascancercelllinesareused.Comparedwithalternativeresources,ourlibraryoffersvari-ousadvantages.First,thefocusednatureofthelibraryenableseffectivescreening,evenwhencomplexreadouts,suchasclo-nogenicorinvasionassays,ormicroscopy-basedmeasurementsareused.Whileapooledgenome-widelibraryrequires200millioncellstocoveritscomplexity1000times[],only3millioncellsareenoughtosaturatethescreenwithourfocusedlibrary.Similarly,forarrayedscreens,theuseofour Figure4.ApplicationsofthearrayedsgRNAlibraryA.Outlineoftheprotocolforlarge-scalegeneeditingandsubsequentfunctionalassays..Quantitativeimmuno-uorescencemicroscopyofcellsinwhichKOofhadbeeninducedfor14dandcontrolcellsnotexpressingsgRNAs(control).isamemberoftheMSLcomplex,whichspecicallyacetylatesH4K16.Forquantication,n=218and123forcontrolandKO,respectively.value:MannWhitneytest.Scalebar:10.Viabilityassaycomparinguninduced(NT)andinduced(DOX)cellsexpressing-targetingsgRNAsandinducibleCas9.n=3.Threeasterisksindicate(Studentt-test)..TumorigenicityassaysinNSGmicecomparinguninduced(NT)andinduced(DOX)cellsexpressing-or-targetingsgRNAsandinducibleCa
s9.n=4.Thenaltumorvolumerelativetothecorrespondinguninducedconditionisindicatedinthegraph.Tumorswereharvested11weeksafterinjection.Twoandthreeasterisksindicate0.05and0.001,respectively. T.HENSER-BROWNHILLETAL. libraryreducesthenumberofrequiredplatesfrom38396-wellplates[]to5whenusingthegenepools,orto33whenusingtheindividualsgRNAs.Second,thepresenceof7sgRNAsformosttargetedgenesincreasesthecondenceintheresultsandminimizestherateoffalse-positivescomparedwithgenome-widearrayedlibrariescontainingonly2sgRNAspergene[whilethedesignofguideswithdistinctstrengths(targeting5ofthegeneoroptimizedactivity)maximizesoverallefThird,beingclonedintoalentiviralvectorthatcanbeeasilypropagated,thegeneratedlibraryisacost-effectivealternativetosyntheticarrayedlibraries[].Fourth,theabilitytotempo-rallycontrolgeneKOwhencombinedwithinducibleCas9ena-blestheuseofsgRNAsinvivo,animportantbenetwhenusinggraftmodels.Inconclusion,thegeneratedCRISPRlibraryrepresentsauniqueresourcetostudyepigeneticmechanismsinvariouscontexts,allowingbothforwardandreversegeneticloss-of-functionscreensandtargetedinvestigationofselectedproteinsinvitroinvivoMaterialsandmethodsCelllinesInvitro-transformedbroblasts[]wereculturedinminimalessentialmedia(MEM)with15%fetalbovineserum(FBS),HepG2cellswereculturedinMEMwith10%FBS,andHEK-293TcellswereculturedinDulbeccosModiedEagleMedium(DMEM)with10%FBS.Mediawassupplementedwith2mML-glutamine,100U/mlpenicillin,and100streptomycin.Tofreezecellsin96-wellformat,cellsweredetachedfromtheplateusing30loftrypsinperwell.Follow-ingadditionof70lofFBScontaining10%DMSO,platesweresealedandstoredat-80Cforupto4weeks.Tothawcells,plateswereplacedinawaterbathat37Cforafewsec-ondsandspunfor5minat4Cafteradditionof50lofmediumtoeachwell.Freshmedium(100l)wasnallyaddedtoeachwellafterremovalof120loffreezingmedium.Forgenerationcelllinesexpressing3xFLAG-taggedinduc-ibleCas9,transformedbroblastsandHepG2cellsweretrans-ducedwithalentiviralpCW-Cas9vector[].(Addgene),whichwasmodiedtoconferresistancetohygromycintoinfectedcells.Followinga7-dayselectionwith600hygromycin,cellswereplatedataverylowdensity(200cells/15cmplate)andgrownfor23weeks.CloneswithminimalbackgroundlevelsofCas9andresponsivetoinductionwith1g/mLdoxycyclinewereidentiedbybothRT-qPCRofCas9expressionandwesternblottingof3xFLAG-tag(SigmaF1804).TogeneratecelllinesstablyexpressingsgRNAs,cellsweretransducedwithpLenti_BSD_sgRNAconstructsandselectedwithblasticidin(4g/ml)for5days.ToinducegeneKO,Cas9expressionwasinducedbytheadditionofdoxycycline(1LibrarydesignsgRNAselectionWherepossible,sgRNAsweredesignedtotargettherstsharedCDSexonofidentiedgenes(RefSeq).Themost5exonofRefSeqvariant1wasutilizedifitwasnotpossibletotargetasharedexongivenaspecicscoringthreshold.Forlateralign-ments,theRefSeqvariant1wasusedasareference.GenomicCDSsequencesseparatedintoindividualexonsassociatedwithRefSeqaccessionsforallgenetargetswereextractedusingthebiomaRtpackageforfor37].CDSsequenceswereextractedmanuallyfromUCSCgenomebrowserin13caseswherethebiomaRtdatabasecouldnotidentifytheinputRefSeqacces-sion.GRCh37/hg19(February2009assembly)wasusedasthereferencegenomethroughoutthedesignstage.FivesgRNAspergene(HensersgRNAgroups15)wereevaluatedforoff-targetactivityusingtheZhanglabspubliclyavailabletoolatcrispr.mit.edu(accessedbetweenAprilandOctober2015).Guideswereselectedusingascorethresholdofatleast70wherepossible,withguidesscoringatleast50usedasbackups.Foreachgeneinthelibrary,2additionalguideswereselectedfromtheCRISPRlibraryconstructedbyWangetal.(WangsgRNAgroups67)[],assuringthatselectedsequencesdidnotoverlapwithexistingguidesbyatleast15bp.ThesgRNAdesignbyWangetal.isbasedonobservedactivityofsgRNAstargetingessentialribosomalgenesindepletionscreens.BriesgRNAswithhighpredictedactivityweredesignedusingasup-port-vector-machineclassierthatidentiedupto80binaryfeaturesofefcientguides[].Overall,ourresulting
librarycontained3150uniquesgRNAsequenceswith7sgRNAspertargetgene.A5nucleotideiscommonlyaddedtosgRNAsexpressedunderaU6promoterforefcientRNApolymeraseIIItranscription;thisadditionalstepwasnotrequiredinthiscaseasa5nucleotideisprependedtoallprotospacersequencesautomaticallyinthevectorduringcloning.cacypredictionOn-targetefcacyofsgRNAswaspredictedinsilicousingbothRulesSet2(RS2)fromtheAzimuthproject[],andsgRNAScorer2.0(SS2)[]predictionmodelsusingavailablepythonscripts[RS2v1.2(accessedAugust2016),SS2v2(accessedMay2017)].InthecaseofRS2onlytherawsequenceoption(--seqwasusedinscorecalculationsintheformatof:NNNN20merNGGNNN.ThepercentageofpeptideremainingfollowingaDSBwaspredictedbymappingsgRNAsequencesagainstcanonicalmRNAsequences,assumingaframe-shiftornonsensemutationwouldbeinducedatthenalnucleotidebeforethePAMsequence.Large-scalesgRNAcloningLentiviralvectorThelentiviralvectorusedtoconstructthissgRNAlibrary(pLenti_BSD_sgRNA)wasgeneratedthroughmodicationofthepLent_sgRNA_LibplasmidutilizedinpreviouslypublishedCRISPR/Cas9screens[],whichweobtainedthroughAddg-ene.TheoriginalGFPreporterwasreplacedwiththeblasticidinresistancecodingregion(BSD)usingGeneArtSeamlessClon-ingandAssembly.LibraryoligosynthesisandannealingForwardandreverseoligonucleotidescorrespondingtothetar-getsequencesweretaggedwith5overhangsappropriatefor-basedcloningstrategy.Single-strandedEPIGENETICS complementaryforwardandreverseoligonucleotidesweresyn-thesizedat100Mina96-wellarrayedformatbySigmaresultingin66(233)96-wellplates.Togeneratedouble-strandedoligonucleotides,relevantpairsweremixedandaddedtoannealingbuffer(10mMTrispH7.5,1mMEDTA,50mMNaCl)usingtheBiomekFXautomatedliquidhandlingequipment,maintainingthe96-wellarrayedformat(SupplementaryTable3).Platesofmixedoligos(x33)werethenannealedinthermocyclersbyheatingthereactionto95for5minsbeforereducingthetemperatureto25Cat0.1Cpersecond.Followingthereaction,annealedoligonucleotideswerediluted1:200beforeproceedingtoGoldenGatecloning.CloningandtransformationCloningofdouble-strandedoligonucleotidesintopLenti-BSD-sgRNAwasperformedusingasingle-stepGoldenGatecloningprocedure(SupplementaryFig.1B).Annealedanddilutedoli-gonucleotidesfromthepreviousstepwereaddedtoeachwellofafresh96-wellPCRplatemaintainingtheoriginalarrayedformatandcombinedwiththecloningmix(SupplementaryTable3).ReactionPCRplateswereplacedinthermocyclersandthecloningreactionperformedoverthecourseofusingtheconditionsindicatedinSupplementaryFig.1.Allplateswerestoredat-20Cuntiltransformation.ChemicallycompetentE.coliStbl3withatransformationciencyof1.5gDNAweregeneratedusingtheMix&GoE.coliTransformationKit(ZymoResearch).TotransformthesgRNAlibrary,competentbacteriawerethawedand50laddedtoeachwellof96-welldeepwellblocks.Subse-quently,2lofconstructsgeneratedin96-wellPCRplatesdur-ingthecloningstepweretransformeddirectlyintothesewells.Alltransformationstepswereconductedonice.CircleGrowmedium(950l;MPBiomedicals)wasaddedtoeachwell,andblockswereinitiallyincubatedovernight(ON)for20hatC.Individualglycerolstocksweregenerated(3396-wellplates).AllindividualglycerolstocksweredigitallytaggedwithDataMatrix2DbarcodesforsimpliedtracingandstoredatGenerationoflibrarypoolsTogeneratethewholelibrarypool,200lofculturefromwellsgrownONweretransferredintothecorrespondingwellsofnewdeep-wellblocks,and800loffreshmediumaddedtoeach.Thenewdeep-wellblockswereculturedforafurther3.5htoequalizetheiropticaldensity(OD)andrespectiveplas-midyields(SupplementaryFig.1D).Atotalof200loftheequalizedculturefromeachwellwerethenpooledgenerating700mlofcultureusedtoextractplasmidDNA.Togenerategenepools,50lofequalizedculturefromeachwellcorre-spondingtothesamegenewerepooledtogetherandusedtogenerateglycerolstocks(5plates,450wells,witheachwellcon-tainingallsgRNAstargetingthesamegene).Viralproductionandmultiplicityofinfection(MOI)estimationTooptimizeconditionsforviraltran
sduction,viruswaspro-ducedinparallelin10cmdish-and96-wellformat.For10cmdish-format,lentiviralparticleswereproducedbytransfectingHEK293Tpackagingcellsat80%conuencewith5gofthesgRNAlentiviralconstruct,3.75gofpsPax2packagingplas-mid,1.25gofpMD2Genvelopeplasmidand1gpAdVant-age(PromegaE1711)ataratioof3:1DNAtoFugeneHD(PromegaE2311).For96-wellformat,thenumberofcellsandtheamountofplasmidDNAwasreduced40times.Approxi-mately24hand48haftertransfection,thesupernatantcon-tainingviralparticleswasrecovered,lteredthrougha0.45lter(MilliporeSLHV033RSorMSHVS4510),pooledtogether,andfrozenat-80C.Inordertodeterminethemultiplicityofinfection(MOI)ofthevirus,1broblastswereseededperwellofa6-wellplateandinfectedwithserialdilutionsofvirusgeneratedin10cmdish-format,startingfrom1:1.Follow-ingselectionwith4g/mLblasticidin,thenumberofviablecells/wellweremeasuredandcomparedtoanon-infectedcon-troltocalculatethepercentageofinfectedcells.TheformulaP=1-e,wherePispercentageofinfectedcellsandmrepre-sentsMOI,wasusedtodeterminetheviralconcentrationyield-ingthedesiredMOIin96-wellformat(typicallyMOI=1).IndividualsgRNAvalidationInitialassessmentofsgRNAactivitywasperformedbytrans-ducingindividualsgRNAsintobroblastscontainingadoxycy-cline-inducibleCas9.Cells(2)wereplatedin10cmdishesandinfectedwithpLenti_BSD_sgRNAlentivirusfor24h.Transducedcellswereselectedwithblasticidinfor4days,afterwhichCas9wasinducedfor12weeks.KOwasassessedviaFACS,quantitativeimmunouorescencemicroscopy,and/orTOPO-cloningandSangersequencingofsgRNAtargetgenes.ForTOPOsequencing,primersweredesignedtoamplifygenomicDNAsequencessurroundingpredictedsgRNAtargetsites(SupplementaryTable4).PCRwasperformedongenomicDNAfromcellsexpressingCas9andrespectivesgRNAs,andampliconsclonedintoblunt-endTOPOvectors.ResultantTOPO-vectorsweretransformedintocompetentE.coliandDNAextractedforsequencing.StandardM13sequencingprimerswereusedtosequencetheclonedamplicons.ForFACSanalysisacelllinetransducedwithemptypTRIPZ(OpenBiosystems)andexpressingTurboRFPwasused.FACSanalysisandimmunouorescenceanalysiswasperformedaspreviouslydescribed[].Antibodiesusedwereanti-H1.0(Upstate)andanti-H4K16Ac(CellSignalingTechnology).cationofuorescenceintensitywasperformedusingMetamorphsoftware.Large-scalesgRNAvalidationToassesstheefcacyonalargerscale,individualsgRNAssepa-ratepoolsweregeneratedforsgRNAgroups5,6,and7,result-ingin3pools,eachcontainingasinglesgRNAforeachofthe450targetgenes.HepG2cellscontainingadoxycyclineinduc-ibleCas9wereplatedat0.45cells/wellina6-wellplateandwereinducedfor24hourspriortoinfectionwiththeabovepoolsinduplicate.CellswereinfectedwithvirusatMOI24hoursafterinfection,cellsweretransferredinto60mmdishesandselectedwithblasticidinfor4days.Doxycyclinewasreplacedevery2daysuntilharvestingofgenomicDNAforsequencing5dayspost-infection. T.HENSER-BROWNHILLETAL. NextgenerationsequencingToassesstherepresentationofindividualsgRNAsinthewholelibraryandinthepools,librariesofampliconscontainingthesgRNAsequencesweregeneratedbyPCRusingtheHerculaseIIpolymerasekit(Agilent).Briey,150ngofthepooledplasmidlibrarieswereusedtogenerate268bp-longamplicons,whichincludedthesgRNAsequenceandtheP5andP7adaptorsforIlluminasequencing.Allprimerswereannealedatatemperatureof60Cfor30secondsandthereactionperformedfor30cycles.Allreactionswereperformedintechnicaltriplicatesandtheirproductspooledandrunona2%agarosegeltoconrmasinglebandof268bp.ThebandsweresubsequentlycutfromthegelsandpuriedusingthegelpuricationKit(Qiagen)aspermanu-facturersprotocol.PuriedproductsweresequencedwithaHiSeq2500usingcustomsequencingandindexingprimers(SeqPandIndexP,SupplementaryTable4).Followingsampledemultiplexing,allsgRNAsequencesweretrimmedandalignedtothetargetsequencestoassesssgRNArepresentation(normal-izedreadcount).Todetectin/delsattargetedregions,librariesweregeneratedusingtheSureSelectTargetenrichmentkit(Agi-lent)usingcustomprobesan
dfollowingthemanufacturerinstructions.Probesweredesignedtocover2kbregionscen-teredoneachtargetsite.Whenmultipletargetsiteswerelocatedinthesameexon,the2kbregionwascenteredontheexonmid-dlepoint.Probetilingparameterswere:Tilingdensity:1x;Mask-ing:LeastStringent;Boosting:MaximizePerformance.Large-scalein/delanalysisThequalityofthesequencedreadswasassuredusingFastQChttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/AlignmentwascarriedoutagainsttheUCSChg19/GRCh37genomeassembly,usingBBMap(v.36.59)(http://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbmap-guide/),aglobalalignerthatisabletoalignlongerin/dels.Atwo-stagealignmentstrategywasusedtorobustlyidentifyreadsthatcon-tainedin/dels.Intherstphase,readswerealignedtothegenomedisallowinganyreadsthatcontainedin/dels.Inthisphase,readsthatalignedinaproperpair(uneditedorcorrectlyrepairedsequences)werediscardedandtheremainderwastakenforward.Inthesecondphase,theremainingreadswerealignedtothegenome,allowingin/delsupto2000bp.Dupli-catesweremarkedusingPicard(v.2.1.0).Readsthatweremarkedasduplicates,orthathadamappingqualityscoreoflessthan38werelteredusingsamtools(v.1.2)[]andsam-bamba(v.0.6.0)[].Thistwo-phaseapproachwasnecessarytodiscriminatebetweenbackgroundnoiseandsignalinourdatasetsinceinfectionwithpooledsgRNAsreturnedahighproportionofuneditedsequencesforeachtargetsequence(onlyasmallfractionofcellswastransducedwitheachsgRNA).AllsubsequentdownstreamanalysisofthereadswasperformedinR(v.3.3.2).Thelocationandsizeofin/delsinreadswereidentiedfromtheCIGARstring.In/delswereonlyconsideredvalidiftheyoccurredwithin2nucleotidesoftheCas9cleavagesite(denedas6nucleotidesupstreamoftheendoftheguideRNAincludingthePAMsequence).Anyin/delsthatcouldalsobedetectedintheuntransducedHepG2samplewereremovedfromtheanalysis.Tocalculatetheobservedactivityscore(OAS),thenumberofin/delsobservedforeachsgRNAwasnormalizedtoitsrepresentationinthepools:themeannumberofobservedin/delsacrosstwobiologi-calreplicates(ID)wasdividedbythenormalizedreadcountforthatsgRNA[rawreadsforasinglesgRNA(sgR)dividedbytotalreadsofallsgRNAsinthepool(TR) ðÞViabilityandtumorigenicityassaysToassessthefunctionalconsequencesofCRISPR-mediatedKOinducedbysgRNAsinourlibrary,transformedhumanbro-blastsexpressinginducibleCas9weretransducedwithsgRNAsagainsteitherINO80(apoolof7sgRNAs)orHRAS(1sgRNA).Followingselectionofinfectedcellswithblasticidin,theresultingpolyclonalpopulationswereusedforphenotypicvalidation.Forviabilityassays,cellstransducedwithINO80-targetingsgRNAswereseededintriplicatesat100,000cells/wellina6-wellplateandculturedinthepresenceorabsenceofdoxycycline(1mL)for8days.Thenalnumberofcells/wellwasdeterminedbymanualcellcountinginthepresenceofTrypanBluetodis-carddeadcells.Forfunctionalinvivoassays,5trans-broblaststransducedwithINO80-targetingsgRNAsandexpressinginducibleCas9wereinjectedintradermallytogetherwith1carrierhTERT-immortalizedbroblastsin50lofPBSintobothanksof68weekoldmaleNOD/SCID/Interleukin2receptornull(NSG)mice.After3weeks,whentumorsbecamepalpable,micewererandomlygroupedandadministeredeither2mg/mLdoxin1%sucrosewateror1%sucrosealone.Sixweeksaftertreatment,tumorvolumewasmeasuredaccordingtotheformuladD/2,wheredandDaretheshortestandthelongestdiameterofeachtumor,respectively.CellsexpressingHRAS-targetingsgRNAswereusedasacontrol.AnimalstudieswereconductedinaccordancewiththeCrickprojectlicensePPL70/8167approvedbytheHomeOfStatisticalanalysesAlldatavisualizationandstatisticalanalyseswereconductedusingthepackagesfor,orMSExcel.Thetestusedforeachanalysisisindicatedinthegurelegend.DataavailabilityThedatasetsgeneratedduringand/oranalyzedduringthecur-rentstudyareavailablefromthecorrespondingauthoronrea-sonablerequestDisclosurestatementTheauthorshavenocompetingnancialinterest.WethanktheCrickcorefacilitiesfortechnicalsupport,particularlyGra-hamClarkandMichaelHowell,andAnobChak
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