Download Highly parallel genome variant engineering with CRISPR

bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder.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.
HighlyparallelgenomevariantengineeringwithCRISPR/Cas9ineukaryoticcells
MeruJ.Sadhu1,*,†,JoshuaS.Bloom1,*,†,LauraDay1,JakeJ.Siegel1,‡,SriramKosuri2,
LeonidKruglyak1,†
1DepartmentofHumanGenetics,DepartmentofBiologicalChemistry,Howard
HughesMedicalInstitute,UniversityofCalifornia,LosAngeles,LosAngeles,CA
90095,USA.
2DepartmentofChemistryandBiochemistry,UniversityofCalifornia,LosAngeles,
LosAngeles,CA90095,USA.
*Theseauthorscontributedequallytothiswork.
†Correspondingauthor.Email:[email protected](M.J.S.);
[email protected](J.S.B.);[email protected](L.K.)
‡Presentaddress:DepartmentofChemistry,MassachusettsInstituteofTechnology,
Cambridge,MA02139,USA.
Abstract:
DirectmeasurementoffunctionaleffectsofDNAsequencevariantsthroughouta
genomeisamajorchallenge.WedevelopedamethodthatusesCRISPR/Cas9to
engineermanyspecificvariantsofinterestinparallelinthebuddingyeast
Saccharomycescerevisiae,andtoscreenthemforfunctionaleffects.Weusedthe
methodtoexaminethefunctionalconsequencesofprematureterminationcodons
(PTCs)atdifferentlocationswithinallannotatedessentialgenesinyeast.Wefound
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder.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.
thatmostPTCswerehighlydeleteriousunlesstheyoccurredclosetotheC-terminal
endanddidnotinterruptanannotatedproteindomain.Surprisingly,wediscovered
thatsomeputativelyessentialgenesaredispensable,whileothershavelarge
dispensableregions.Thisapproachcanbeusedtoprofiletheeffectsoflargeclasses
ofvariantsinahigh-throughputmanner.
Maintext:
UnderstandingthefunctionaleffectsofDNAsequencevariantsisofcritical
importanceforstudiesofbasicbiology,evolution,andmedicalgenetics,but
measuringtheseeffectsinahigh-throughputmannerisamajorchallenge.One
promisingavenueispreciseeditingwiththeCRISPR/Cas9system,whichallows
generationofDNAdouble-strandbreaks(DSBs)atgenomicsitesmatchingthe
targetingsequenceofaguideRNA(gRNA).RecentstudieshaveusedCRISPR
librariestogeneratemanyframeshiftmutationsgenome-widethroughfaultyrepair
ofCRISPR-directedbreaksbynonhomologousend-joining(NHEJ)(1).Wesoughtto
adaptthisapproachtoprecisevariantengineering.Generationofprecisegeneedits
byCRISPR/Cas9requiresintroductionofarepairtemplatethatcanbeusedto
directrepairthroughhomology-directedrepair(HDR)pathways(2),intheprocess
incorporatingthedesiredsequencevariantspresentonthetemplateintothe
genomiclocus.Generatingmanyuniquelyeditedcellsinparallelthusrequireseach
celltoreceivethecorrectgRNA-repairtemplatepair.Wedevisedanapproachthat
accomplishessuchpairingbyencodinggRNAtargetingsequencesandtheir
correspondingrepairtemplatesincisonoligonucleotidesgeneratedinbulkwith
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
high-throughputsynthesis.Theseoligonucleotidelibrariesarethenusedto
generatepoolsofplasmidspairingthetwocomponentsfordeliveryintoyeastcells.
Asimilarmethodwasrecentlyreportedinbacteria(3).Weusedthisapproachto
understandtheconsequencesofoneimportantclassofgeneticvariants:premature
terminationcodons(PTCs).
PTCsinterrupttheopenreadingframes(ORFs)ofprotein-codinggenes.Such
mutationsaregenerallyexpectedtohavestrongdeleteriouseffects,eitherby
abrogatingorchangingthefunctionsoftheencodedproteinsorbycausingmRNA
degradationthroughthenonsense-mediateddecay(NMD)surveillancepathway.
Morethan10%ofannotatedpathogenichumanvariantsarePTCs(4,5).
Nonetheless,ourunderstandingofthedetrimentaleffectsofPTCsisincomplete,
particularlywhentheyoccurnearthe3’endsofgenes.Suchmutationsmaynot
shortentheencodedproteinssufficientlytoaffecttheirfunction,andoftenescape
NMD.
Wefirsttestedgeneeditingthatemploysaplasmid-encodedpairedgRNAand
repairtemplate(figure1a)bytargetingeightspecificPTCstotheS.cerevisiae
genome.S.cerevisiaehasanaturallyhighpropensitytorepairDSBsthroughHDR
(6),whichweenhancedbyusingayeaststraininwhichNHEJisabolishedbya
deletionoftheNEJ1gene(7)(SupplementaryTable1).Foreachtargetedmutation,
wesequencedthecorrespondinggenomiclocusinthousandsoftransformedyeast
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
cells.Inalleightcases,thedesiredmutationwaspresentin>95%ofsequencing
reads,demonstratingthehighefficiencyofthisstrategy(Table1).
Wenextscaleduptheapproachbyusinglarge-scaleoligonucleotidesynthesisto
generateapoolofover10,000distinctpairedgRNA-repairtemplateplasmids
(Supplementaryfigure1).TheseplasmidstargetedPTCstodifferentsitesin1034
yeastgenesconsideredessentialforviability(8,9).Eachgenewastargetedat10
sites,chosenwithapreferenceforsitesclosertothe3’end(SupplementaryFigure
2).Wetransformedyeastinbulkwiththisplasmidpool.AfterinducingCas9
expression,wecollectedmillionsofsurvivingtransformedcellsevery24hoursfor
fourdays(figure1b).PTCsthatdisruptthefunctionofgenesessentialforviability
areexpectedtodropoutofthepoolovertime,whilethosethatdonotareexpected
topersist.
Wedeterminedtheabundanceofeachedit-directingplasmidateachtimepointby
bulksequencing,andcomputeda“PTCtolerancescore”basedonthepersistenceof
eachplasmidoverthedurationofthetime-courseexperiment(Materialsand
Methods).Ascontrols,weusedasetof90“dubiousORFs,”whichwereoriginally
annotatedasgenesbutlaterreclassifiedduetolackofconservationandascribable
function(10).Asexpected,PTCsinessentialgenesweremuchlesstoleratedthan
thoseindubiousORFs(Wilcoxonranktest,P<2x10-16)(figure1c).Asafurther
control,71sitesinessentialgenesweretargetedwithtwoplasmidsthathadthe
samegRNAbutdifferentrepairtemplates,onlyoneofwhichintroducedaPTC.
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
PlasmidsthatintroducedaPTCweresignificantlylesstolerated(Supplementary
Figure3)(pairedt-testt=6.5,P=8x10-9),showingthattheobservedphenotypic
effectsarepredominantlyduetospecificintroductionofthedesiredmutations,
ratherthanrepair-template-independentCas9activities.
OnepossibilityfortheobservedPTCintoleranceisthatmosttruncationsof
essentialgenesfatallydisruptproteinfunction.AnotherpossibilityisthatNMD
removesmosttranscriptscarryingPTCs,whichisfatalinthecaseofPTCsin
essentialgenes.WetestedthesealternativesbyintroducingPTCsinastrainthatis
NMD-deficient(11).PTCsinthisstrainweresimilarlydeleterious(Supplementary
Figure4)(χ2=1.66,P=0.20)(SupplementaryTable8),suggestingthatprotein
truncation,ratherthandegradationoftranscriptsviaNMD,explainstheobserved
PTCintolerance.
AlthoughmostPTCsinannotatedessentialgeneswerehighlydeleterious,some
appearedtobetolerated.Weexaminedtherelationshipbetweentolerancescores
andlocationsofPTCs.PTCsweregenerallydeleteriouswhenlocatedmorethan27
codonsawayfromthegeneend(figure1d).Withinthe27terminalcodons,the
tolerancescoresrosetowardthe3’end.PTCswerealsomoretoleratediftheydid
notinterruptorremoveanannotatedproteindomain(12)(χ2=317.2,P=5.86x1071)(Supplementaryfigure5,SupplementaryTable8).PTCsthatdisruptedprotein
domainstendedtobedeleteriousevenwhentheyfellclosetogeneends.
Evolutionaryconservationofthetruncatedregionamongrelatedyeastspecies(13)
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
wasalsosignificantlybutmoreweaklypredictiveofPTCtolerance(χ2=49.8,P=
1.66x10-12)(Supplementaryfigure5).
Webuiltamodeltomorepreciselydelineatedispensable3’endsofessentialgenes.
Whileourexperimentisnotdesignedtocomprehensivelyruleouttheexistenceof
smalldispensableC-termini,itisinterestingtonotethat517genesdidnotappear
totolerateanytestedPTCs(Supplementaryfigure6),insomecasesevenveryclose
totheirends.Forinstance,weconfirmedthatPob3,involvedinnucleosome
remodelingduringDNAreplicationandtranscription,doesnottoleratelossofits
lasttwoaminoacids(SupplementaryFigure6),whichareadjacenttothenuclear
localizationsequence(14).WealsoconfirmedthatPCNA,requiredforthe
processivityofDNApolymerase,didnottoleratethelossofitslastfiveaminoacids.
ThishighlyconservedregionispartofthebindingsurfaceofPCNAusedforproteinproteininteractions(15).IncontrasttothehighlyPTC-intolerantgenes,101genes
toleratedfiveormorePTCs,suggestingthatthesegeneshavelargedispensableCtermini(SupplementaryFigure6).WecomputedtheoveralltoleranceofPTCsfor
eachgeneandobservedconsiderablevariationamonggenes(figure2a).Agene
ontologyenrichmentanalysis(16)showedthatgenesencodingproteinswith
catalyticactivityweresignificantlylessPTC-tolerantthanothergenes(KolmogorovSmirnovtest,BonferronicorrectedP=0.0024)(Supplementarytable10,
SupplementaryFigure7),whilegeneswithfunctionsrelatingtomRNAsplicingand
processingweresignificantlymorePTC-tolerant(Kolmogorov-Smirnovtest,
BonferronicorrectedP=0.0017).
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
Tobetterunderstandwhysomegenesannotatedasessentialcouldtoleratemany
PTCs,wecloselyexaminedthe16mostPTC-tolerantgenes(figure2a).Thesegenes
includedSSY1,PTR3,andSSY5,thethreemembersoftheSPS(Ssy1-Ptr3-Ssy5)
plasmamembraneaminoacidsensorsystem(17),aswellasSHR3,requiredforSPS
cell-surfacelocalization(18).DefectsinSPSfunctioncompromiseleucineuptake,
andthestrainoriginallyusedtodeterminewhichgenesareessentialisdeficientin
leucinebiosynthesisandthusrequiresleucineuptake,whichexplainsthelethality
ofSPSmutationsinthisstrain(19,20).Weconfirmedthatdeletionsofthesegenes
wereviableinyeastthatcouldsynthesizeleucine,butlethalinyeastthatcouldnot
(figure2b).Similarly,thePTC-tolerantgeneFUR1isrequiredfortheutilizationof
exogenousuracil(21),anduracilbiosynthesisisalsodisruptedinthestrainusedto
annotateessentialgenes.WeconfirmedthatFUR1isonlyessentialinyeastwhich
cannotsynthesizeuracil(figure2b),consistentwithprevioussyntheticlethality
results(22).Unexpectedly,wealsoobservedpoorgrowthofyeastwithdeletionsof
bothURA3andthePTC-tolerantgeneSDH3(figure2b),amemberofthe
mitochondrialinnermembraneproteintranslocasecomplex(23),whichsuggests
thatproperuracilutilizationmayinvolveanunknownmitochondrialfunction.
Theseexamplesillustratethatgenesnotuniversallyessentialforyeastviabilitycan
appearessentialinaspecificgeneticbackground.
AnotherPTC-tolerantgene,MMF1,encodesaproteinhomologoustothewidely
conservedRidA,whichprocessestoxiciminesproducedduringisoleucine
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
biosynthesisinSalmonella(24).WenoticedthatdeletionofMMF1isviableunder
ourgrowthconditions,butnotunderthosepreviouslyusedtodefinethesetof
essentialgenes(Supplementaryfigure8).Thusinthiscase,thelethalityofagene
disruptionisdeterminedbyagene-environmentinteraction.Wehypothesizethat
MMF1isessentialinyeastwhenisoleucineissynthesized,butnotwhenitistaken
upfromthegrowthmedium.Importantly,theisoleucinetransportersBap2and
Bap3arepoorlyexpressedunderthegrowthconditionsusedtoannotateessential
genes,butmorehighlyexpressedunderthegrowthconditionsusedinour
experiments(25).
ThreeadditionalPTC-tolerantgenesweremisannotatedasessentialbecausetheir
deletiondisruptsthefunctionofanearbyessentialgene.YJR012Coverlapsthe5’
endofthePTC-intolerantessentialgeneGPI14(Supplementaryfigure9,figure2a).
CloseexaminationofYJR012CRNAsequencingandribosomefootprintingdata(26)
indicatesthatthestartpositionofYJR012Cismisannotated(Supplementaryfigure
9).DeletionofYJR012CfromitstruestartatM76totheendoftheORFwasviable,
confirmingYJR012Cisnotessential.Similarly,closeexaminationofUTR5revealed
thatitoverlapstheTATAboxoftheessentialgeneHYP2(27);deletionfromthe
34thcodonofUTR5sparedtheTATAboxandwasviable(Supplementaryfigure9).
Finally,deletionofTRE2isinviableduetoitseffectsontheneighboringessential
geneCDC31(28).TheseexamplesillustratethevalueofPTCintroductionfor
characterizationofgeneessentiality.
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
SixPTC-tolerantessentialgenesencodeproteinswithlargedispensableC-terminal
regions.OnestrikingcaseisCWC24,ahighlyconservedmemberofthespliceosome.
Cwc24hasaCCCH-typeZincfingerdomain(Znf)andaRING-typeZnfdomain.
AnalysisoftheeffectofPTCsinCWC24suggestedtheRINGfingerdomainwas
dispensablewhiletheCCCHZnfwasessential(figure3a),whichweconfirmedby
engineeringCWC24truncations(seealsoWuetal.,2016(29)).Itisinterestingto
notethataPTCaftertheRINGfingerdomainoftheessential(30)humanhomologof
CWC24,RNF113A,isviable(31).FourotherPTC-tolerantgenes,TAF7,TAF8,COG3,
andLSM4,havebeenreportedtotoleratelargetruncations(32–35).Weverifiedthat
SEC5,a971-aminoacidmemberoftheessentialexocystcomplex(36),tolerates
truncationofatleast615aminoacids(figure3b).Ourobservationthat101genes
toleratedfiveormorePTCssuggeststhatmanyadditionalgeneshavedispensable
C-terminalregions.
Ourresultsimprovetheannotationofessentialgenesinthewell-studiedyeast
genome.Wediscoveredseveralcasesofgenesthatappearedtobeessentialasa
consequenceofthespecificstrainandgrowthconditionsoriginallyusedtotest
viabilityofgenedeletions.Adeletionscreeninadifferentyeastisolatealsorevealed
examplesofconditionallyessentialgenes(37).Applyingourapproachandrelated
methods(38)inadiversesetofisolatesandgrowthconditionswillfurtherrefine
thecoresetofessentialyeastgenes.
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
PTCsareprioritizedinstudiesofhumangeneticsbecauseofthehighlikelihoodthat
theyabolishgenefunction.OurresultssuggestthatPTCsaremostlikelytobe
deleteriouswhentheydisruptannotatedproteindomainsortruncatemorethan27
aminoacids,andthesecriteriamayimprovefilteringofcandidatecausalvariants.
WeobservedthatNMDdidnotmakeastrongcontributiontoPTCtolerance.This
resultisconsistentwithrecentfindingsthatNMDinyeastactsmoststronglyon
transcriptswithPTCstowardtheir5’ends(39).PTCsneartheendsofhumangenes
arealsolikelytoescapeNMDaccordingtothe50-base-pairrule(40)
(Supplementaryfigure10),andourcriteriamaybeespeciallyusefulforpredicting
theireffects.
Inourstudywecarriedoutapooledscreenofthefunctionaleffectsof
approximately10,000directedmutations.Ourmethodcanbeusedtoassessthe
functionaleffectsofanydesirednucleotidevariantsinahighlyparallelmanner.The
abilitytoprofiletheimpactofbroadclassesofalleles,includingmissenseand
regulatoryvariants,willenableamorefine-grainedunderstandingofthe
relationshipbetweengenotypesandphenotypes.
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Acknowledgements:
WethankKruglyaklaboratorymembers,F.Albert,M.P.Hughes,andJ.Rinefor
helpfuldiscussion,R.CheungandE.Phamfortechnicalassistance,andG.Churchfor
plasmids.FundingwasprovidedbytheHowardHughesMedicalInstituteandNIH
grantsR01GM102308(L.K.)andF32GM116318(M.J.S.).
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
Figures:
Figure1:MeasuringtheeffectsofengineeredPTCsinessentialgenes.(A)Schematic
ofpairingofCRISPRgRNAandrepairtemplateonplasmids.(B)Experimental
design.FollowingCas9induction,DNAwasextractedevery24hours.Ateachtime
point,edit-directingplasmidswerequantifiedbysequencing.(C)Tolerancescore
foreachtestedPTCinessentialgenesanddubiousORFs,withoverlaidboxplots.P<
2x10-16,Wilcoxonranktest.(D)ScatterplotofPTCtolerancescoresversusdistance
incodonsfromthe3’endsofessentialgenes.Thethickbluelineshowsasegmented
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
regressionfit.Verticalbluelinesindicatethe95%confidenceintervalforthe
boundarybetweenthesegments.
Figure2:PTCtoleranceofgenes.(A)Genetolerancescoresforessentialgenesand
dubiousORFs,shownasaviolinplotthatdisplaystheindividualdatapoints.(B)
Analysisofconditionallyessentialgenesinyeasttetrads.Eachverticalsetoffour
coloniescorrespondstothefourhaploidmeioticproductsfromadiploidyeast
strain.Eachdiploidwasheterozygousforadeletionmutationofinterestandforan
interactingmutation.Haploidcoloniescarryingthedeletionofinterestare
highlightedinredorbluebasedontheirgenotypeattheinteractinglocus.Absence
ofavisiblecolony(firstfivepanels)indicatesalethalinteraction;smallcolonies
(lastpanel)indicateaninteractioncausingpoorgrowth.
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
Figure3:Selectedtruncatableessentialgenes.(A)Tolerancescoresfor10PTCsin
CWC24areshownbygraycircles;redandgreenbarsindicateHMMcallsof
‘deleterious’and‘tolerated’,respectively.TheRINGfingerandCCCHZnfdomainsof
Cwc24arehighlighted.(B)Analysisofdeleteriousandtoleratedtruncationsof
CWC24inyeasttetrads,displayedasinfigure2.Deletionsofthelast88and94
codonsofCWC24aretolerated(middleandrightpanels),whiledeletionofthelast
119codonsisnot(leftpanel).(C)TolerancescoresforeightPTCsinSEC5areshown
bygraycircles;redandgreenbarsindicateHMMcallsof‘deleterious’and
bioRxiv preprint first posted online Jun. 8, 2017; doi: http://dx.doi.org/10.1101/147637. 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.
‘tolerated’,respectively.ThePfam-annotated“SEC5domain”ishighlighted.(D)
AnalysisofdeleteriousandtoleratedtruncationsofSEC5inyeasttetrads.Deletion
ofthelast615codonsofSEC5istolerated(rightpanel),whiledeletionofthelast
707codonsisnot(leftpanel).
Tables:
Expectededit
Unedited
Mismatch
Indel
ho-G582Stop
98.51%
0.08%
1.40%
0.00%
his2-E308Stop
99.83%
0.07%
0.10%
0.00%
mnd1-V219Stop 99.35%
0.53%
0.12%
0.00%
spo11-F381Stop 95.56%
4.24%
0.19%
0.00%
spo13-P252Stop 99.67%
0.20%
0.13%
0.00%
ste3-P469Stop
99.75%
0.12%
0.13%
0.00%
can1-G121Stop
99.81%
0.06%
0.13%
0.00%
can1-G70Stop
99.80%
0.03%
0.17%
0.00%
Table1:Assessingtheefficiencyofedit-directingplasmids.Outcomesofdirected
mutationsateightlociinnej1∆cells,asdeterminedbypaired-endIlluminareadsof
PCRsofgenomicDNAateachlocus.