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bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. CRISPR-directedmitoticrecombinationenablesgeneticmappingwithoutcrosses MeruJ.Sadhu*,JoshuaS.Bloom*,LauraDay,LeonidKruglyak* DepartmentofHumanGenetics,DepartmentofBiologicalChemistry,andHoward HughesMedicalInstitute,UniversityofCalifornia,LosAngeles,LosAngeles,CA90095, USA *Correspondenceto:[email protected](M.J.S.),[email protected] (J.S.B.),[email protected](L.K.) Abstract Linkageandassociationstudieshavemappedthousandsofgenomicregionsthat contributetophenotypicvariation,butnarrowingtheseregionstotheunderlyingcausal genesandvariantshasprovenmuchmorechallenging.Resolutionofgeneticmappingis limitedbytherecombinationrate.WedevelopedamethodthatusesCRISPRtobuild mappingpanelswithtargetedrecombinationevents.Wetestedthemethodby generatingapanelwithrecombinationeventsspacedalongayeastchromosomearm, mappingtraitvariation,andthentargetingahighdensityofrecombinationeventsto theregionofinterest.Usingthisapproach,wefine-mappedmanganesesensitivitytoa singlepolymorphisminthetransporterPmr1.Targetingrecombinationeventsto bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. regionsofinterestallowsustorapidlyandsystematicallyidentifycausalvariants underlyingtraitdifferences. IdentificationofDNAsequencedifferencesthatunderlietraitvariationisacentralgoal ofmoderngeneticresearch.Theprimarytoolsforconnectinggenotypeandphenotype arelinkageandassociationstudies.Inthesestudies,co-inheritanceofgeneticmarkers withthetraitofinterestinlargepanelsofindividualsisusedtolocalizevariantsthat influencethetraittospecificregionsofthegenome.Thelocalizationreliesonmeiotic recombinationeventsthatbreakuplinkagebetweenmarkersonachromosome. Therefore,thespatialresolutionofgeneticmappingislimitedbytherecombination rate.Inpractice,therecombinationrateinmostsettingsistoolowtoresolvethe mappedregionstoindividualgenes,muchlesstospecificvariantswithingenes. Increasingmappingresolutionrequiresconstructionofever-largerpanelsofindividuals and/oradditionalgenerationsofrecombination,andtheseapproachesarelaboriousto thepointofoftenbeingimpractical.Asaconsequence,thegenesandvariants underlyingtraitvariationhaveyettobeidentifiedforthevastmajorityofregions implicatedbylinkageorassociationmapping. Toaddressthisproblem,wehavedevisedanewmethodforgeneticmappingthat preciselytargetsrecombinationeventstoregionsofinterest.Themethoduses recombinationeventsthatoccurduringmitosisratherthanmeiosis.Raremitotic recombinationeventsoccurnaturallywhenachromosomaldoublestrandbreak(DSB)is bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. repairedbyhomologousrecombination(HR)thatleadstotheformationofa recombinedchromosome(YinandPetes2013).Inaheterozygousindividual,mitotic celldivisioncanthengeneratedaughtercellswithanewgenotypethatiscompletely homozygousfromtherecombinationsitetothetelomereandunchangedheterozygous everywhereelse(Fig.1A);suchaneventistermed“lossofheterozygosity”(LOH). IndividualswithLOHeventsatvariouslocationsinthegenomehavebeenusedto constructageneticmap(Hensonetal.1991),andthisandrelatedapproaches(Laureau etal.2016)can,inprinciple,beusedtomapthegeneticbasisoftraitvariation(Fig.1B). However,thisapproachhasbeenlimitedinpracticebytheverylowfrequencyof naturalmitoticrecombinationevents. WehaveleveragedtheCRISPR-Cas9systemtoproducetargetedmitoticrecombination eventsathighfrequencyandatanydesiredlocation,allowingfacileconstructionof LOH-basedmappingpanels.IntheCRISPR(clustered,regularlyinterspaced,short palindromicrepeats)system,thebacterialendonucleaseCas9isdirectedtocreatea DSBatasitespecifiedbyavariabletargetingsequenceofaboundguideRNA(gRNA) (DoudnaandCharpentier2014).Successfulcuttingalsorequiresthetargetedsequence tobefollowedbyaninvariantprotospacer-adjacentmotif(PAM).AnLOHeventina heterozygousdiploidindividualcanbegeneratedbycuttingonlyonechromosome, leavingitshomologintacttoserveasatemplateforrepairbyHR.Thisisaccomplished byusingpolymorphicheterozygousPAMsitesthatarepresentononlyoneofthetwo chromosomes. bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. InordertodemonstratethatLOHeventscanbetargetedtoprecisegenomiclociusing CRISPR,wedesigned95gRNAstargetingStreptococcuspyogenesCas9tosites distributedacrosstheleftarmofS.cerevisiaechromosome7(Chr7L).ThegRNAs targetedheterozygoussitesinadiploidyeaststraingeneratedbycrossingalabstrain (BY)andavineyardstrain(RM),usingPAMspolymorphicbetweenthetwostrains.After cutting,repair,andmitosis,cellsinwhichtheDSBrepairledtoanLOHeventwere isolatedbyfluorescence-activatedcellsorting(FACS)throughtheirlossofatelomereproximalgreenfluorescentprotein(GFP)gene.WepickedapproximatelyfourGFP(-) linespertargetedsite,foratotalof384lines.Genotypingbylow-coveragewholegenomesequencingdemonstratedthatCRISPR-inducedrecombinationwashighly effective,withLOHeventsinmorethan95%oflinesandfewoff-targeteffects (SupplementaryMethods).MostLOHrecombinationeventsoccurredwithin20kbof thetargetedcutsite(Fig.2),consistentwithpreviousmeasurementsofLOHgene conversiontractlength(St.CharlesandPetes2013).LOHeventsweresuccessfully generatedatsitesacrosstheentirelengthofthetargetedchromosomearm(Fig.2), demonstratingthatourmethodisnotlimitedtocertaingenomiccontexts. WenextusedtheLOHpaneltomapquantitativetraitstolocionChr7L.Wemeasured growthofeachofthe384LOHlinesin12differentconditions,chosenbecausewe previouslymappedquantitativetraitloci(QTLs)forgrowthintheseconditionstoChr7L. Inparallel,wemeasuredgrowthof768segregantsfromacrossbetweenBYandRM bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. (Bloometal.2013).Oneofthetraits,growthon10mMmanganesesulfate,mappedto alarge-effectQTLwithamaximumLODscoreof109.4intheLOHpanel(Fig.3).The confidenceintervalobtainedwiththe384LOHlinesoverlappedwithandwasnarrower (2.9kb)thanthatobtainedwith768segregants(3.9kb).TheLOH-basedinterval containedtwogenesand12polymorphismsbetweenBYandRM.Weidentified concordantQTLsofsmallereffectforeightothertraitsinthetwopanels(fig.S1).Two traitsmappedtoaQTLofsmalleffectsizeinjustonepanel,likelyduetolowstatistical power(fig.S2).OnetraitlackedaChr7LQTLinbothpanels. Torapidlyfine-mapthecausalvariantformanganesesensitivity,wegeneratedasecond panelofLOHlineswhoserecombinationeventswerealltargetedtothemapped manganesesensitivityinterval.WetookadvantageofthefactthatLOHgeneconversion tractsvaryinlength,whichmeansthatindifferentindividuals,DSBsgeneratedbythe samegRNAcanleadtoslightlydifferentLOHcrossoversites,typicallywithin10kbof theDSB(St.CharlesandPetes2013).Weisolated358GFP(-)linesgeneratedwiththree gRNAstargetingsitesnearthemappedinterval.Wegenotypedthispanelbysequencing andobservedthat46ofthelines(13.1%)hadarecombinationeventwithinthe2.9kb QTLinterval;together,therecombinationeventsseparatedalmostallthevariantsinthe interval(Fig.4A).Incontrast,only0.7%ofsegregantshadrecombinationeventsinthe interval(Bloometal.2013).Toobtainacomparablenumberofrecombinationeventsat thislocusbyrandommeioticsegregation,asegregantpanelwouldrequiremorethan bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 7,500lines.Thus,withtargetedLOHevents,wecanineffectgenerateunnaturally strongrecombinationhotspotsatanyregionofinterest(fig.S3). Wemeasuredmanganesesensitivityinthisfine-mappingpanel(Fig.4B).Comparisonof thepanelphenotypeswiththebreakpointlocationspinpointedasinglepolymorphism asresponsibleforincreasedsensitivityinBY.Thepolymorphismchangesa phenylalanineinBYtoaleucineinRMatposition548ofPmr1,amanganese transporter.SixlineshadrecombinationeventsbetweenPmr1-F548Landtheclosest polymorphismtotheright,402bpaway,andwereeitherfullysensitiveorresistantto manganese,dependingonwhichPmr1-F548Lallelewashomozygousintheline.One linehadarecombinationbetweenPmr1-F548Landtheclosestpolymorphismtothe left,125bpaway,andshowedtheintermediatemanganesesensitivityphenotype expectedforaheterozygoteatthecausalvariant.LODscoreanalysisofthefinemappingpanelalsoidentifiedasupportintervalcontainingonlyPmr1-F548L(Fig.4B). TodirectlytesttheeffectofPmr1variantsonmanganesesensitivity,weindividually engineeredtheRMallelesofPmr1-F548L,thetwoneighboringpolymorphisms,aswell asthetworemainingnonsynonymousPmr1polymorphismsintoBY.Variant replacementswerecarriedoutinasinglestepbyCRISPR-directedDSBformation combinedwithrepairoffasuppliedtemplatecarryingthedesiredallele.Asexpected basedontheLOHfine-mapping,changingphenylalanine-548toleucinemadeBY bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. significantlymoreresistanttomanganese,whereasnoneoftheotherfour polymorphismsqueriedhadasignificanteffectonmanganesesensitivity(Fig.5). PMR1encodesanionpumpthattransportsmanganeseandcalciumintotheGolgi (Culottaetal.2005).Pmr1isamemberoftheP-typeATPasefamilyofionandlipid pumpsthatarefoundinallbranchesoflife,andmanyotherP-typeATPaseshavea conservedleucineatthepositionhomologoustophenylalanine-548ofPmr1.TheNMR structureoftheratsodiumpump(Hilgeetal.2003)andthecrystalstructureofthe rabbitcalciumpump(ToyoshimaandMizutani2004)showaleucineatthehomologous positionmakingdirectcontactwithATP(fig.S4).Furthermore,mutatingthe homologousleucineoftherabbitcalciumpumptophenylalaninedecreasesfunctionby affectingATPbinding(Clausenetal.2003).Thus,theF548Lpolymorphismisexpected toreducetheabilityofPmr1BYtotransportmanganeseintotheGolgi,relativeto Pmr1RM,consistentwiththeobservedmanganesesensitivityofBY. Pmr1leucine-548isconservedacrossfungi,withafewspecieshavinganisoleucineor valineatthehomologousposition,andnonehavingphenylalanine(fig.S5).IntheS. cerevisiaepopulation,almostallsequencedPMR1alleleshaveleucine-548,with phenylalanine-548foundonlyinBYandotherlaboratoryyeaststrains(Litietal.2009; Songetal.2015)whosePMR1allelesarelikelydirectlyrelatedtoBY(Schachereretal. 2009).ThisregionofBYChr7isinheritedfromEM93,adiploidyeaststrainisolated fromafiginCaliforniain1938(MortimerandJohnston1986).SequencingofPMR1in bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. EM93revealedthatEM93isheterozygousforPmr1-F548L(fig.S6),suggestingeither thatthemutationisnotlaboratory-derivedorthatitoccurredbetweentheisolationof anditsentryintoastockcollection. Decadesofmappingstudieshaveuncoveredlociformyriadtraits,butidentificationof theunderlyinggenesandvariantshaslagged.WedevelopedanewCRISPR-assisted mappingapproachthatpromisestoclosethisgap.OurapproachusesCRISPRto generatetargetedrecombinationeventsdenselycoveringaregionofinterestofany size.WefirstgeneratedCRISPR-directedLOHeventsthatspanayeastchromosome arm,andusedtheresultinglinestomaptraitvariation.WethensaturatedaQTL intervalwithrecombinationeventstorapidlyidentifyasinglecausalpolymorphism responsibleformanganesesensitivityinlaboratorystrainsofS.cerevisiae.We confirmedthecausalroleofthispolymorphismbydirectCRISPR-assistedengineeringof theresistancealleleintoasensitivestrain.Incontrasttopreviousstrategies,our methodgeneratesamuchhigherdensityofrecombinationevents,iseasilytargetableto anyregionofthegenome,anddoesnotrequireextratime-consuminggenerationsof crossingtoincreaserecombinationfrequency. WeanticipatethattraitmappingwithtargetedLOHpanelswillgreatlyaideffortsto understandthegeneticbasisoftraitvariation.Inadditiontomanyapplicationsinsinglecelledorganisms,LOHpanelscouldbegeneratedfromculturedcells,enablinginvitro geneticdissectionofhumantraitswithcellularphenotypes.Mappingresolutionin bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. multicellularorganismscouldbeenhancedwithCRISPR-directedmeioticrecombination events.Indeed,themutagenicchainreactionsystemdevelopedinvivoinfruitflies (GantzandBier2015)andmosquitos(Gantzetal.2015;Hammondetal.2015)uses CRISPRtogenerategeneconversioneventsinmeiosiswithextremelyhighefficiency. AnotherapproachwouldbetostimulateLOHduringearlydevelopment,generating chimericindividuals.AnotherapproachwouldbetostimulateLOHearlyin development,generatingchimericindividuals.ThetargetedLOHmethodalsohasthe excitingpotentialtobeappliedtoviableinterspecieshybridsthatcannotproduce offspring,allowingtraitvariationbetweenspeciestobestudiedgeneticallybeyondthe fewsystemswhereitiscurrentlypossible(OrrandPresgraves2000;Woodruffetal. 2010). Recently,severalgroupshavereportedusingCRISPRtocreatechromosomal rearrangements(ChoiandMeyerson2014;Maddaloetal.2014;Lietal.2015).Tothe bestofourknowledge,oursisthefirstreportofusingCRISPRtogenerateLOHevents. Inadditiontotheirresearchapplications,targetableendonucleasesholdpromisefor genetherapy(Hsuetal.2014;Tebasetal.2014).Certaindiseaseallelesmaybevery difficulttodirectlytargetbyCRISPRbecauseoftheirsequencecomplexity;oneexample isprovidedbytheexpandedtrinucleotiderepeatsthatunderlieHuntington’sdisease.In thesecases,directingaDSBtooccurinthevicinityofapathogenicallelesothatitis replacedwithitsnonpathogeniccounterpartbyLOHmayrepresentamorefeasible alternative. bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. Acknowledgements WethankthemembersoftheKruglyaklaboratoryforhelpfuldiscussionandcritical readingofthemanuscript,andGeorgeChurchforplasmids.Fundingwasprovidedby theHowardHughesMedicalInstituteandNIHgrantR01GM102308(L.K.). bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. Fig.1:DSBsgeneratedbyCas9indiploidmitoticcellscanleadtomitoticrecombination andlossofheterozygosity(LOH).(A)LOHcanresultfromrepairfollowingadoublestrandbreak(DSB)inmitosis,whichisgeneratedbyCRISPR.IndividualswithLOHevents areisolatedviathelossofaheterozygousdominantmarker,denotedwithanasterisk (*).(B)BymeasuringtraitvaluesinapanelofindividualswithLOHeventsdistributed acrossaregionofinterest,wecanmapgeneticvariantsthatcontributetotrait variation.Theprocesscanbeiteratedtogainextremelyfinemappingresolution. bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 300 kb 200 kb 0 100 kb Observed LOH recombination position 400 kb cen 500 kb cen 0 100 kb 200 kb 300 kb 400 kb 500 kb Position targeted for DSB Fig.2:ObservedLOHrecombinationlocationvs.siteofDSBtarget.Foreachindividualin thepanelwithaChr7Lrecombinationevent,thesiteofitsrecombinationeventis plottedagainstthesitetargetedforDSBformationinthatindividual.Individuals targetedtogainBYandRMhomozygosityareplottedinorangeandpurple, respectively.Thedashedlinesencloseindividualswithrecombinationeventswithin20 kbofthetargetedsite. 60 50 100 40 80 20 20 40 30 LOD 60 10 0 growth on 10 mM Manganese Sulfate (colony radius) bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 0 100kb 200kb 300kb Chr 7 position 400kb 500kb Fig.3:Sensitivitytomanganesevs.observedLOHrecombinationlocation.Foreach individualintheChr7Lpanel,thesiteoftheLOHrecombinationeventisplottedagainst manganesesensitivity,measuredascolonyradiusaftergrowthon10mMmanganese sulfateplates.OrangeandpurplepointsdenoteindividualsthatarehomozygousBYand RMtotheleftoftheirrecombinationevents,respectively.(Allindividualsare heterozygousBY/RMtotherightoftheirrecombinationevents.)Thegraylineplotsthe LODscorebypositionalongChr7Lformanganesesensitivity.Dashedverticallines denotetheQTLsupportinterval. bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 4 0 2 cM/kb 6 8 A 190 kb Chr 7 position 195 kb 100 35 150 40 185 kb 50 30 LOD BY/RM polymorphism 0 25 B growth on 10 mM Manganese Sulfate (colony radius) 180 kb ||| | || ||| | || 186 kb || || | || || | || 188 kb || | || | | || 190 kb Chr 7 position | | | | 192 kb | | || | 194 kb || Fig.4:Targetedhigh-resolutionmappingofmanganesesensitivity.(A)Ratioof recombinationrate(incentimorgans;cM)tophysicaldistancenearthemanganese sensitivityQTL,forthemanganesefine-mappingLOHpanel(black)andasegregant panel(red)(Bloometal.2013).cM/kbisshownforintervalsbetweenBY/RM polymorphismsthatareatleast300bpapart;thefine-mappingpanelcontains bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. recombinationeventsbetweenallsuchpairsofpolymorphismsintheinterval,ascM/kb doesnotdroptozero.The2.9kbQTLintervaldeterminedbythewhole-Chr7LLOH panelisdenotedwithdashedlines.(B)Recombinationsitesofindividualsinthefinemappingpanelplottedagainsttheirmanganesesensitivity,asinFigure3,nearthe manganesesensitivityQTL.ShownbelowareallBY/RMpolymorphismsintheregion,as wellasallopenreadingframes.DashedbluelinesdenotetheQTLsupportintervalfor thefine-mappingpanel;forreference,dashedblacklinesdenotetheQTLsupport intervalforthewhole-Chr7Lpanel. 30 35 40 * 25 growth on 10 mM Manganese Sulfate (colony radius) 45 bioRxiv preprint first posted online Feb. 19, 2016; doi: http://dx.doi.org/10.1101/040428. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 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