Download CRISPR-directed mitotic recombination enables genetic

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 The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder.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 The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder.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
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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.
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LOD
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growth on 10 mM Manganese Sulfate (colony radius)
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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.
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
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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
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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.
BY
RM
A46T
I187L
L414M
F548L
BY with Pmr1 site-directed mutations
A589 (Syn)
Fig.5:DirectintroductionofPmr1-F548LintoBYenhancesmanganeseresistance.
BoxplotsofmanganesesensitivityforstrainswithsinglePMR1variantsintroducedfrom
RMintoBY,alongwiththeBYandRMparentalstrains.n≥10forallgenotypes.*p<
0.001incomparisontoBY.
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
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