Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9

20 Pages·2016·5.46 MB·English

RESEARCHARTICLE
Manipulating the Prion Protein Gene
Sequence and Expression Levels with
CRISPR/Cas9
LechKaczmarczyk1,YlvaMende2,BrankoZevnik2,WalkerS.Jackson1*
1 GermanCenterforNeurodegenerativeDiseases(DZNE),Bonn,Germany,2 CologneExcellenceCluster
onCellularStressResponsesinAging-associatedDiseases(CECAD),MedicalFaculty,Universityof
Cologne,Cologne,Germany
a11111 *[email protected]
Abstract
Themammalianprionprotein(PrP,encodedbyPrnp)ismostinfamousforitscentralrolein
priondiseases,invariablyfatalneurodegenerativediseasesaffectinghumans,foodani-
OPENACCESS
mals,andanimalsinthewild.However,PrPisalsohypothesizedtobeanimportantrecep-
Citation:KaczmarczykL,MendeY,ZevnikB,
torfortoxicproteinconformersinAlzheimer'sdisease,andisassociatedwithother
JacksonWS(2016)ManipulatingthePrionProtein
GeneSequenceandExpressionLevelswith clinicallyrelevantprocessessuchascancerandstroke.Thus,keyinsightsintoimportant
CRISPR/Cas9.PLoSONE11(4):e0154604. clinicalareas,aswellasintounderstandingPrPfunctionsinnormalphysiology,canbe
doi:10.1371/journal.pone.0154604
obtainedfromstudyingtransgenicmousemodelsandcellculturesystems.However,the
Editor:JiyanMa,VanAndelInstitute,UNITED Prnplocusisdifficulttomanipulatebyhomologousrecombination,makingmodificationsof
STATES
theendogenouslocusrarelyattempted.Fortunatelyinrecentyearsgenomeengineering
Received:March22,2016 technologies,likeTALENsorCRISPR/Cas9(CC9),havebroughtexceptionalnewpossibili-
Accepted:April16,2016 tiesformanipulatingPrnp.Herein,wepresentourobservationsmadeduringsystematic
experimentswiththeCC9systemtargetingtheendogenousmousePrnplocus,toeither
Published:April29,2016
modifysequencesortoboostPrPexpressionusingCC9-basedsynergisticactivationmedi-
Copyright:©2016Kaczmarczyketal.Thisisan
ators(SAMs).Itisourhopethatthisinformationwillaidandencourageresearchersto
openaccessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits implementgene-targetingtechniquesintotheirresearchprogram.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Introduction
Funding:Thisworkwassupportedbyinternal
PrPisanextracellular,membrane-anchoredglycoprotein,abundantlyexpressedinbrainofa
fundingfromtheDZNE.
widerangeofspecies,especiallymammals[1].ThefunctionofthephysiologicalformofPrP,
CompetingInterests:Theauthorshavedeclared calledthecellularformorPrPCforhistoricalreasons,isamatterofdebate,butevidenceexists
thatnocompetinginterestsexist.
foritsroleinsynapticplasticity[2–4],cellsignaling[5],andneuroprotection[6–10],especially
Abbreviations:Cas9,CRISPR-associatedsystem fromstroke[11,12].PrPCcanbesubvertedintoneurodegenerativecascadesviabindingto
nuclease9;CC9,CRISPR/Cas9;CRISPR,clustered toxicproteinassembliesrelatedtoAlzheimer'sdisease[13–15].Inagroupofrareneurological
regularlyinterspacedshortpalindromicrepeats;DSB,
disordersknownaspriondiseases(PrDs)PrPistwistedintoatoxicconformer–PrPsc–whichis
doublestrandbreak;ESCs,embryonicstemcells;
alsoaninfectiousagent(prion)[16,17].Althoughrare,PrDshavemanyfeaturesincommon
HR,homologousrecombination;NHEJ,non-
homologousendjoining;Prnp,prionproteingene; withmoreprevalentneurodegenerativediseases,likeAlzheimer’sorParkinson’sdiseases[18–
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ManipulatingPrnpwithCRISPR/Cas9
PrP,prionprotein;RIT,randomintegrationtransgenic; 20].AremarkablefeatureofPrDsisthatasingleprotein,PrP,cancauseprofoundlydifferent
SAM(s),synergisticactivationmediator(s);sgRNA, diseases[21–23].Thus,factorsandmechanismsleadingtodifferencesinneurodegenerative
shortguideRNA;TALENs,transcriptionactivator-like
diseasesingeneralcanbeinvestigatedbystudyingPrDs.KeyinsightsintoPrPbiologicalfunc-
effectornucleases;ZFNs,zincfingernucleases.
tionswereobtainedfromstudiesoftransgenicmiceexpressingPrPmutants.Forexample,
mousestudiesconclusivelyshowedthatPrPCisabsolutelyrequiredforPrPsctoxicity[24,25]
andthatsequencechangesinPrPlargelyaccountfordifferencesinvulnerabilitytoprioninfec-
tion[26,27].However,mechanismsofprionreplicationandtoxicityremainelusive,likely
involvingacomplexinterplaybetweenphysiologicalpropertiesofindividualcells,theamount
andsequenceofPrPexpressed,andthenatureandsequenceofinvadingprions[28].
MuchcanbelearnedfromcreatingmousemodelsexpressingPrPvariants.Suchmodels
canbeengineeredviagene-targetingoftheendogenousgenelocus(Prnp)toproduceknock-in
mice(KIs),orbyallowingPrPexpressingtransgenestointegrateintorandomlocationsinthe
genome,resultinginrandomintegrationtransgenicmice(RITs).Animportantadvantageof
theRITapproach,asidefromtherelativeeaseofthetechnique,isincreasedexpressionofPrP,
acceleratingthediseaseprogressionandexacerbatingtheeffectsofmutations[29–31]thatare
typicallymuchmilderwhenexpressedfromtheendogenouslocation[22,32,33].
However,RITsarevulnerabletopositioneffectsleadingtodisparatespatialexpressionpat-
ternsindifferentlines,aconfounderthatisoftenignoredeventhoughitlikelycontributesto
phenotypicdifferencesbetweendifferentmutantsexpressedasRITs[34].Unfortunately,
manipulationoftheendogenousPrnplocusinmicehasbeenachallenge.Thefirstattemptto
knockoutPrnpinvolvedselecting,isolating,andscreeningof~10.000embryonicstemcell
(ESC)clonesbytediousSouthernblotting[35,36].Despitesomesignificantimprovements
[32,37–39],thelowefficiencyofmodifyingPrnpinESCswithhomologousrecombination
(HR)haslongthwartedeffortswiththisprocedure,mostlikelyduetoa“closed”chromatin
stateofthelocus[40–42].Toavoidthisdifficulty,mostinvestigatorscontinuetodevelopPrP
transgenicmicewithRITs,oftendrivenbythemoPrP.XhoIvectorcomposedoffragmentsof
Prnpfrommultiplesources[43–45].NewtoolstomanipulateendogenousPrnpweresorely
neededtoreducethetechnologicalhurdlesincreatingPrnpKImice.
Helpcamewiththeadventofpowerfulnewgenomeengineeringtechnologies,especially
TALENsandCRISPR/Cas9(CC9),whichbroughtunprecedentedprecisionandefficiencyto
targetedmutagenesis,makinggenerationofgeneKIseasierandfaster[34,46–48].Sincethe
CC9systemistheoreticallyverysimpletoprogramtotargetspecificlocationsbymodifying
theshortsingleguideRNA(sgRNA)component(Fig1),wetesteditsefficiencyinstimulating
HRinthePrnplocusinmouseESCs.WeevaluatedseveralsgRNAsandidentifiedsomethat
werehighlyefficientinmutatingESCsandmousefertilizedoocytes.Furthermore,weevaluated
severalsgRNAsascomponentsofSAMcomplexes(seeFig1,leftpanel)fortheirefficiencyin
stimulatingPrnpexpression[49],obtainingupto10-foldincreasesinmRNAandproteinlev-
els.ImprovedPrnptargetingwillgreatlyfacilitateproductionofmoreconsistentdiseasemod-
els,andPrnp-directedSAMscancomplementexistingmodelsbyacceleratingPrPexpression
ubiquitously,orinspecificcellpopulations.
MaterialsandMethods
Ethicsstatement
ExperimentswereapprovedbytheStateOfficeofNorthRhine-Westphalia,Departmentof
Nature,EnvironmentandConsumerism(LANUVNRW),approvalnumber84–02.04.2014.
A372.Allmeasuresweretakentominimizethenumberofanimalsused,aswellastomaxi-
mallyreducetheirdiscomfortandpain.
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ManipulatingPrnpwithCRISPR/Cas9
Fig1.TwoapproachesinvolvingCRISPR/Cas9forgenemanipulation.Ontheright,aCas9nucleasetogetherwithsgRNA(inred)inducesadouble
strandbreak(DSB)intheDNA.DamageresultsinrecruitmentofDNArepairmechanisms,includinghomology-mediatedrepair(HDR),whichcanbe
harnessedtointroduceand/orreplaceDNAfragments.Ontheleft,aSAMcomplexconsistingofcatalyticallyinactiveCas9(dCas9)fusionwithVP64
activationdomainandsgRNA2.0(inblue)bindstoapromoterregionandstimulategeneexpression.Transcriptionisfurtheraugmentedbyrecruitmentofa
proteinfusioncomprisingp-65andheatshockfactor1(HSF1)transcriptionfactorsandMS2bacteriophagecoatprotein,whichassociatethecomplexto
MS2stem-loopsinsgRNA2.0[49,66].
doi:10.1371/journal.pone.0154604.g001
Animals
FertilemaleandfemalemicetogeneratefertilizedoocytesweresuppliedbyCharlesRiver
(France).Fosterfemalesandmalesformakingthempseudopregnant(bothRjHan:NMRI)
weresourcedfromJanvierlabs(France).Animalswerekeptina12hlight/12hdarkphotope-
riodat22°C(±2°C)andahumidityof55%(±5%)withaccesstowaterandfoodadlibitum.
MicemaintenanceandhandlingwasaccordingtolocalgovernmentregulationsandtheDirec-
tive2010/63/EUrevisingDirective86/609/EEC).
Vectorgeneration
Prnptargetingvectorsweregeneratedusingstandardmolecularbiologytechniques.Aconven-
tionalPrnpKO(tagging)vectorwasconstructedsimilartoapreviousone[38]exceptthatthe
HPRTmini-genewasbluntligatedintopWJPrP38[32]openedbydigestionwithEagIand
ClaIrestrictionendonucleases,therebyspecificallyremovingPrPproteincodingsequencebut
leavingtherestofexon3intact.KItargetingconstructsweregeneratedwithatwo-stepprocess.
First,aneomycinselectioncassetteflankedbyFlprecombinasesites(Addgeneplasmid
#22687)wasligatedintoanengineeredEcoRVsitelocated78bpupstreamofPrnpexon3in
thepWJPrP38targetingvectortemplate[32],creatingpWJPrP101.Second,PrPORFvariants
(hamsterandbankvolePrPvariants,andPrP-EGFPfusionproteins)wereligatedintotheClaI
andEagIsitesofpWJPrP101.DetaileddescriptionsofKIvectorswillbeincludedinreports
describingthespecificmouselines.SpacersequencestogeneratepX330(Addgeneplasmid
#42230)andpX459(Addgeneplasmid#62988)derivativesfortargetingPrnplocuswere
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ManipulatingPrnpwithCRISPR/Cas9
sourcedas5’phosphorylatedoligonucleotides(SigmaAldrich),annealedthroughcycleofheat-
ingandslowcoolingin50mMNaCl,dilutedto100nM,andclonedintoBbsIsite.Vectorsfor
SAMswereAddgeneplasmidsplasmids#61422(MS2–p65–HSF1),#61423(dCas9-VP-64),
#61424(sgRNA2.0backbone)(see[49]).SpacerswereclonedintosgRNA2.0backbonesame
wayasincaseofpX330andpX459.
EScellculturing,targetingandgenotyping
MouseV6.5EScells[50]weregrownongelatin-coatedplates,onaconfluentlayerofPMEF-N
fibroblasts(Millipore),at37°Cin10%C0 ,inDMEMcontaininghigh(4500mg/L)glucose,20%
2
EScell-qualifiedFCS(Millipore),1xnucleosides(Millipore),4mML-glutamine(Gibco),1mM
Na-pyruvate(Gibco),1xNEAA(Gibco),1xPenicillin-Streptomycin(Gibco),7μl/L2-mercap-
toethanoland1000U/mlLIF(Millipore).ESCswerefeddailyandsplitevery2–3days.PMEF-N
fibroblastsweregrowninthesamemediumasESCs,butcontainingstandardFCSandnoLIF
andnucleosides.Forelectroporation,5–10x106cellswereused.ESCsweretrypsinized,gently
trituratedtobreakcellclumpsandsuspendedinESCelectroporationbuffer(Millipore).Cells
wereelectroporatedusingGenePulserXcell(Biorad)setto300V/250μF.40μgofhomology
templateand10μgofCC9vectorswereusedperelectroporation.DNAforelectroporationwas
preparedbythefollowingsteps:1)isolationfromStbl3E.colistrain(Lifetechnologies)using
EndoFreeMAXIPrepKit(Qiagen),2)overnightdigestionof100–200μgDNAwithXhoI
restrictionenzyme(NEB)in400μlreactionvolume(1UofenzymeperμgofDNA,3)precipita-
tionbyadditionof1ml100%EtOH,4)transferringtheprecipitatedDNAusingp1000pipetteto
anewtubecontaining1mlof70%EtOH,5)pelletingandresuspendingin100–200μlofESC
electroporationbuffer(Millipore).Afterthepulse,cellsweretransferredintowarmESCmedium
anddistributedintofeeder-coatedplates.Geneticin(Gibco,350μg/ml)selectionwasstartedafter
18–24hrsandcontinuedfor3dayswithdailychangeoftheselectionmedium.Afterwards,colo-
niesweregrownforadditional4–6daysinESCmedium(untilreadyforharvesting),harvested
with1000μlpipette,trypsinizedandplatedongelatinandfeeder-coated24-wellplates.Clones
wereexpandedfor3–4days,andthenfrozen,leavingsmallaliquotsofcellsfurtherexpandingfor
DNAisolation.Forgenotyping,DNAfromconfluent24-wellplateswasisolatedbylysisinbuffer
containing10mMTris-ClpH7.5,10mMNaCl,10mMEDTA,0.5%SDSand1mg/mlprotein-
aseK,followedbyEtOHprecipitation.LongPCRtoconfirmgenomiclocationwereperformed
withLongAmpTaqpolymerase(NewEnglandBiolabs),using65°Cannealing/extensiontemper-
atureand1min/kbextensiontime.UsedprimersequencesarelistedinS1Table.
Pronuclearinjection(PNI)ofCC9components
ThreedayspriortoPNIfifteen3–4weeksoldC57BL/6NCrlfemaleswereinjectedintraperito-
neal(i.p.)with5IUofPMSG(pregnantmare’sserum),followedbyi.p.injectionof5IUof
hCG(humanChoriongonadotropin)48hlater.Femalesweresubsequentlymated1:1with12
weeksoldC57BL/6NCrlmales.Onthedayofinjectionplugcheckwasperformedandfemales
weresacrificedbycervicaldislocation.Fertilizedoocyteswerecollecteduponpreparingovi-
ductsandrupturingtheampullae.Washingandincubationstepswereperformedasdescribed
elsewhere[51].Theinjectionmix(100ng/μlCas9RNA,50ng/μlsgRNA,20ng/μltargeting
constructDNA)wascentrifugedfor10min/4°C/9838gandplacedonicepriortoinjection.
Injectionsweredirectedtothemalepronucleus.Constantpressurethroughouttheprocessled
toinjectionofsmalleramountsofinjectionmixintothecytoplasm.Unilateralembryotransfer
intopseudopregnantfosterfemaleswaseitherdirectlyperformedorwhenembryosreached
the2-cellstage.DNAfromobtainedfounderswasPCRamplifiedusing5’-ATCACCATCAA
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GCAGCACAC-3’forwardprimerand5’-CAACGTCAGTAGGACAATTTGA-3’reverse
primer,isolatedfromthegelandsequencedwiththeSangermethodusingtheforwardprimer.
N2acellmaintenance,transfectionandFACSsorting
N2acellswereculturedunderthesameconditionsasMEFs.Confluentcellsweretransfected
withcombinationsofvectors:MS2–p65–HSF1,dCas9-VP-64,andsgRNA2.0containingPrnp-
specificsgRNAs(PrnpSAM1–9),withlipofectamine3000accordingtothemanufacturer’s
protocol,usingrespectively15μg/15μl/15μlofDNA/lipofectamine/P3000reagentper10
cmplate(orscaledproportionally).Forfluorescence-activatedcellsorting(FACS)experiments
cellswereharvestedbytrypsinization24hposttransfection,suspendedinMEFmediumata
densityof~5x106cells/mlandkeptonice.EGFP-positivecellswereenrichedbyFACSon
FACSAriaIIIcellsorter(BecktonDickinson),using530/30emissionfilterforEGFP.Cellpop-
ulationsweregatedaspresentedinS1Fig.Sortedcellsuspensionswerechilledduringsorting
and(immediatelyafter)spundownandsnap-frozen(forimmunoblotting)orlysedinRLT
buffer(forsqRT-PCR).
sqRT-PCR
TotalRNAwaspurifiedwithRNeasyPlusMiniKit(Qiagen).FinalRNAconcentrationswere
adjustedtothevalueoftheleastconcentratedsample.Reversetranscriptionwasdonewith
TaqManReverseTranscriptionKit(ABI)usingthemanufacturer’sprotocol,withoutaddition
ofDTT,withamixtureofoligod(T)andrandomhexamersforpriming,and0.5μgoftotal
RNAastemplate.0.5μlofobtainedcDNAwasusedforRT-PCRreaction,usingSYBRGreen
PCRMasterMix(ABI),in384-wellPCRplates(ABI)witha7900HTFastReal-TimePCRSys-
tem(ABI).Foreachreaction,criticalthresholdcycle(Ct)valuewasdeterminedusingSDS5.0
Software(ABI)andprimerspecificitywasconfirmedbymeltcurveanalysis.Twoindependent
primerpairsforPrnpwereused,andthedatawereanalyzedwiththeΔΔCtmethod,usingthe
averageCtvaluesofHPRTandß-actin(ACTB)fornormalization.Primersequences(5’-3’):
Prnp1_F:GTCGCATCGGTGGCAGGACT,Prnp1_R:CAGCCAGTAGCCAAGGTTCGCC,
Prnp2_F:ACATCTGAAGTATGGGACGC,Prnp2_R:TAGGGGTCTGCTTTGGAATC,
HPRT_F:CTCTGGTAGATTGTCGCTTATC,HPRT_R:CTCTTAGATGCTGTTACTGAT
AG,ACTB_F:GTTACAGGAAGTCCCTCACC,ACTB_R:ACCAAAGCCTTCATACAT
CAAGT.
Immunoblotting
Deep-frozencellculturepelletswerelysedin2xLDS(lithiumdodecylsulfate)samplebuffer
containing40mMDTT.Lysateswereforcedthrougha27Gneedleseveraltimestocleave
DNAandreduceviscosity,andthendenaturedat80°Cfor10minpriortoloadingon10%
NuPAGENovexmidigels(LifeTechnologies).GelswererunusingMES[2-(N-morpholino)
ethanesulfonicacid]runningbufferat160V.Gelswereelectro-transferredtonitrocellulose
membrane(Bio-Rad),submergedintransferbuffer(20%methanol,25mMTris-Cl,0.19M
glycine),usingaCriteriontransfertank(BioRad),at700mA,for70minutes.Membraneswere
blockedfor20minatRTin5%powderedmilkinPBST(PBSwith0.05%tween-20)andthen
incubatedwithprimaryantibodydilutedinblockingbufferovernightat4°C.Next,blotswere
washed4xwithPBSTandincubatedwithsecondaryantibodyfor30minatRT,followedby5
PBSTwashesatRTandimagingwiththeLi-CorOdysseysystem.Forre-probing,membranes
werestrippedfor10minwithRe-Blotsolution(EMD-Millipore),followedbyrepeatedblock-
ing.MembraneswereimagedwiththeOdysseyimagingsystem(LI-CORBiosciences).Primary
antibodies:mouseanti-PrPSAF83(dil.1:2000,CaymanChemical),mouseanti-α-tubulin(dil.
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1:10000,SigmaAldrich).Secondaryantibody:donkey-anti-mouseIRDye800CW(dil.1:20000,
Li-Cor).
FACSanalysisandimmunocytochemistryonN2acells
N2acellswereculturedandtransfectedasdescribedabove.ForFACSanalysis,topreservecell
surfacePrP,cellswereharvestedwithouttrypsin,byscrapinginPBS(withoutCa2+andMg2+).
Priortoscraping,cellswereincubatedfor1hwithSAF83primaryantibody(dil.1:2000,Cay-
manChemical),washedwithPBSandincubatedwithAlexa647-coupledgoat-anti-mousesec-
ondaryantibody(LifeTechnologies).PriortoFACS,suspensionswerefilteredthrough70μm
mesh.DatawereacquiredusingtheFACSCantoIIflowcytometer(BDBiosciences)andana-
lyzedwithFlowJosoftwarev.10.1(TriStar,Inc.).Forimmunocytochemistry,cellswerecul-
turedon8-wellchamberedcoverglass(Nunc),stainedidenticallyasforFACSanalysis,and
imagedwithLSM700confocalmicroscope(CarlZeiss).
Statisticalanalysis
GenetargetingexperimentsresultswereassessedwithCochran-Mantel-Haenszeltestfor
repeatedtestsofindependence.Ineachcase,theexperimentwasrepeatedatleastthreetimes,
andatleasteightcolonieswereanalyzedperreplicate.ForsqRT-PCRingeneactivationexperi-
ments,Kruskal-Wallistestwasused,followedbyDunn’smultiplecomparisonpost-test.
Results
EvaluationofCC9sgRNAsformanipulatingthePrnplocus
PriortothediscoveryofCC9,wetypicallyperformedgene-targetingexperimentsusingthe
doublereplacementortagandexchangestrategy[22,32,52,53].Thisstrategyinvolved1)tar-
geting(or“tagging”)thePrnplocuswithaselectablemarker(HPRT),and2)re-targetingthe
locus,thistime“exchanging”thetaggedalleleforaninvitromodifiedallele.TheHPRTmini
geneisanespeciallyusefulselectablefeature:cellswithHPRTactivityareresistanttohypoxan-
thine-aminopterin-thymidine(HAT),whereascellswithoutHPRTactivityareresistantto
6-thioguanine(6-TG)[32].Usingthisstrategy,ourroutinegene-targetingexperimentswere
carriedoutontwotothreereplicatesdoneonsuccessivedays.Oneachday,typicallythree10
cmdisheswereselectedforeachelectroporation,occasionallyresultinginatotalofthreeto
fivepositiveclones,butoftenresultinginnone.SinceESCsrequirefreshmedianearlyevery
day,atacostof~twoEuroper10cmdish,plusthetimetoperformthelabor(whichisinvalu-
able),itbecameasignificantinvestmenttocreatetheselines.Thus,therewasmuchroomfor
improvement.
ForourinitialassessmentofPrnpgenetargetingefficiencywithCC9,weusedthesame
homologytemplatevectoraswithournumerouspreviousgene-targetingexperiments,this
timecontaininga4kbtransgenebetweenthehomologyarms(Fig2A).Wedesignedsixdiffer-
entsgRNAsandtestedeachseparately(seeFig2Aand2BandS2Table).Spacersequences
wereclonedintothepX459CC9vector,carryingexpressioncassettesencodingbothhybrid
sgRNAandspCas9-2A-Puro(Fig2B)[54].Weenvisionedusingpuromycintoselectforcells
expressingthepuromycinresistancegeneandthustheCas9nuclease(co-expressedbythe
spCas9-2A-Puroplasmid).Unfortunately,theexquisitetoxicityofpuromycinonESCsisnoto-
riouslydifficulttotitrateandtheconferredresistancefromthespCas9-2A-Puroplasmidwas
insufficienttorescueourrapidlydividingESCs.Therefore,ourstrategyreliedonG418selec-
tionoftheneomycinresistancegenecontainedintheKItargetingconstruct.Tofacilitatethe
directcomparisonofconventionaltagandexchangetargetingtoCC9directedHR,wecreated
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Fig2.EvaluationofCRISPR/Cas9forPrnplocustargeting.(A)Schematicgeneknock-inintothemousePrnp
locususinghomologyvectorusedforalltargetingexperiments.sgRNAbindingsitedweremarkedwithwhitearrows.
Primerbindingregionsusedforsubsequentgenotypingofrecombinedclonesweremarkedwithpurplearrowsand
designatedp1throughp6.NeoR–neomycinresistancegene;FRT–Flprecombinaserecognitionsiteforremovalof
selectioncassetteaftersuccessfulgenetargeting.Regionsofhomologywereshadedingrey.(B)Schematicofthe
experimentalworkflowforsgRNAevaluation.Inbrief,cellswereelectroporatedwithaPrnptargetingvectoronly,or
PrnptargetingvectorandaCC9encodingguides#1-#6.Ineachcase,targetingefficiencywasassessedbylong
PCRsspanningthehomologyarms.(C)HistogramofCC9-assistedtargetingefficiencyforeachofthesixtested
sgRNAcomparedto“noCC9”control(firstcolumn).“*”—p<0.05;“**”—p<0.01;“***”—p<0.001;Cochran-Mantel-
Haenszeltest.
doi:10.1371/journal.pone.0154604.g002
aheterozygousESClinethatiswild-typeononePrnpallelewhiletheotherallelehadthePrP
codingsequence(Fig2A,"PrPORF")replacedwithanHPRTminigene(PrnpWT/HPRT,not
shown).ThetargetingconstructsincludedPrPsequencesfromdifferentspecies,withnumer-
oussequencedifferencesfromthemousePrPcodingsequence.Asaresult,thetransgenespro-
videdpoorsubstrateforhomologousrecombinationwiththeendogenousPrPcoding
sequence,andthereforethehomologyarmsontheendswouldbetheonlydriversofHR.
Althoughthetargetingconstructscarriedanegativeselectablemarker(HSV-TK),thedrugit
confersresistanceto(gancyclovir)wasnotapplied.Thereby,integrationeventswerenot
selectedagainst,creatingastrategyanalogoustotheearliestgene-targetingexperiments[35].
WiththiscombinationoftargetingconstructandeitherWTorheterozygousESClines,we
weresettodetect,withinthesamegene-targetingexperiment,HReventsdependentonCC9
comparedtothosefromnon-CC9HRevents.Thelatterwouldbeexpectedtobeenrichedin
eventsthatexchangedthePrnpHPRTalleleandconferredresistanceto6-TG.Whentestedin
WTorPrnpWT/HPRTESCs,threeoutofsixguidesledtosignificantimprovementofthetarget-
ingefficacyascomparedwiththecontrolexperimentswherenoCC9wasused(Fig2C).Inter-
estingly,forexperimentswithPrnpWT/HPRTcells,allG418-resistantclonesweresensitiveto
6-TG,indicatingthattheWTallele,andnottheHPRTallelewasreplaced,andthatHRfacili-
tatedbyCC9greatlyexceededHRwithoutCC9.Weselectedguide#3(HRPrnp3,Fig2C)for
furtherexperiments.Usingthesamehomologytemplate,wereproduciblyobtainedHReffi-
cienciesexceedingthoseofrandomintegration(themajorityofG418-resistantcellsweregene-
targeted)(Fig3A).Importantly,applicationofCC9resultedinanearlyten-foldincreaseinthe
proportionofclonesthatwerepositive(79.2±7.2%forHPRT/WTcells)and(88.6±4.6%%)for
WT/WTcells)comparedtoexperimentswithoutCC9(9.7±2.4%,Fig3B).
Althoughwedidnotattempttoquantifythetotalnumberofclones,experimentswithCC9
routinelyyieldsfarmorecoloniesthanourpreviousstrategy(describedatthebeginningofthis
section),enablingustoreducetheoverallnumberofcellsneededforselection,typicallyobtain-
ingmanypositiveclonesevenwhenselectingonasingle6-wellplatewell.Forexample,instead
ofselectingfortargetedclonesonthree10cmdishesofeachelectroporation,wenowtypically
selectonthree6-wellplatewells,eachtypicallyyieldingmorethanfivepositiveclones.Asthe
surfaceareaofa10cmdishis~150cm2andthatfora6-wellplatewellis9cm2,theshiftfrom
usingalargenumber(sixtonine)of10cmdishestoasmallnumberof6-wellplatewells
(threeisenoughtoacquirethreeindependentclones)hasresultedinasignificantreductionin
culturingcosts.Moreover,everyexperimentresultsinalargenumberofcolonies,mostof
whicharepositive,allowingustobehighlyselectivewhenpickingonlymorphologicallyperfect
colonies,withhighconfidencethatthegene-targetingattemptwillnotneedtoberepeated.
EncouragedbythehighHRefficiencyinESCs,wethenattemptedtouseCC9withHRPrnp
3totargettransgenesofvarioussizes(3-7kb)intothePrnplocusinWTEScells.Eachclone
wastestedforcorrectrecombinationwithtwoindependentPCRassays,spanning5’and3’
homologyarms.WeconsistentlyobtainedHRrates>80%forallclonesanalyzed(Table1).In
parallel,weattemptedtoinduceHRbydirectinjectionofallcomponentsintothepronucleus
PLOSONE|DOI:10.1371/journal.pone.0154604 April29,2016 8/20
ManipulatingPrnpwithCRISPR/Cas9
Fig3.TargetingofthePrnplocuswithCRISPR/Cas9.(A)ExamplePCRshowingrecombinationofa4kb
longtransgeneintoPrnplocususingHRPrnp3sgRNA;n.c.–negativecontrol;p.c.–positivecontrol;M—marker
ladder.Reverseprimerinthetoppanelannealstoboth,WTandMUTsequences,andtherebycandiscriminate
betweenhomozygousandheterozygousknock-in;Clone#8wasconfirmedtobehomozygous.(B)Cumulative
CC9targetingefficiencyassessmentofwithHRPrnp3sgRNAinunmodifiedV6.5andV6.5Prnp(WT/HPRT)cells
(containingonlyonecopyofWTPrnpgene).“***”—p<0.001;Cochran-Mantel-Haenszeltest.(C)Fragments
ofsequencingchromatogramsofDNAisolatedfromfoundermiceobtainedbypronuclearinjectionofCC9with
HRPrnp3sgRNA.DNAsequencingoftwooutofthreefoundersrevealedashiftinelectrophoregram
correspondingtoanin-delinthenucleasetargetsiteinoneofthealleles.TherelevantPAMsequencewas
shadedingray.
doi:10.1371/journal.pone.0154604.g003
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ManipulatingPrnpwithCRISPR/Cas9
Table1. GenetargetingefficiencyintothePrnplocususingHRPrnp3spaceroffourdifferenttarget-
ingconstructscontainingvariantsofBankVolePrPandneomycinselectionmarker(FNF).
Targetingconstruct Clonestargeted/analyzed Targetingefficiency
#1 20/22 91%
#2 18/19 95%
#3 16/20 80%
#4 18/19 95%
doi:10.1371/journal.pone.0154604.t001
offertilizedmouseoocytes(PNI).Tothisend,weusedthesamehomologytemplateusedin
theinitialscreenofsgRNAs(4kbfragmentbetweenhomologyarms).FromasinglePNI
attempt,weobtainedonlythreefounderanimals,withnohomologousrecombinantsamongst
them.Nevertheless,PCRsequencingoftheCC9targetsiterevealedin-delsintwooutofthree
founderlines,indicatingCC9withHRPrnp3ishighlyefficientinvivo(Fig3C).
IdentificationofCC9-basedSAMsthatactivatePrnp
Inadditiontoitspowerfulabilitytomodifygenomicsequences,theCC9systemcanalsobe
employedtoinduce[49,55]orrepress[55,56]geneexpression.Multiplestrategieshavebeen
successfullydemonstratedbuttheyallsharethefeaturesofusingaCas9derivativewiththe
nucleaseactivityinactivated(deadCas9ordCas9)andfurthermodifiedtorecruittranscrip-
tionaltransactivationcomponents,suchasthroughinclusionoftheveryrobustVP64transac-
tivationdomain(Fig1).Importantly,thedCas9-VP64fusionproteinisdirectedtospecific
genomiclocationswithsgRNAs,justlikeforexperimentswithnucleaseactiveCas9.Inthesys-
temweemployed,theguideRNAsalsoincludedanextrapairofstemloopsthatrecruitan
additionaltransactivationdrivingfusionprotein,MS2-p65-HSF.Todetermineifwecould
boostmousePrnpexpressionwiththissystem,wedesignedninesgRNAspacerstargetingvari-
ousregionsupstreamofthePrnptranscriptionstartsite(TSS)(Fig4A),withtheaidofan
onlineSAMsgRNAspecificitypredictiontool(http://sam.genome-engineering.org/database/)
andsgRNAscoringalgorithm[57],http://www.broadinstitute.org/rnai/public/analysis-tools/
sgrna-design-v1).Sequencesandscoresofindividualguidesusedinthisstudyareincludedin
S3Table.SinceESCsdivide~threetimesperday,weusedamurineN2aneuroblastomacell
linesincetheydividefarlessfrequentlyandwillallowfortheCC9componentstobeexpressed
andthenpotentiallyregulatePrnp.N2Acellsweretransfectedwithcombinationsofvectors
encoding1)dCas9-VP64fusion,2)MS2–p65–HSF1fusionproteinand3)sgRNA2.0backbone
containingMS2stemloopsandtestgRNAspacers(Fig4B)[49].Components1and2also
containedanEGFPreporter,enablingustoenrichfortransfectedcellsbyFACS(S1Fig).
WhilealltestedsgRNAsledtoanincreaseinPrPRNAlevels,asdeterminedbysqRT-PCR,
sgRNAs#1and#2providedthegreatestenhancement(Fig4C).Thesetwoguideswerefurther
evaluatedinthreeadditionaltransfectionexperiments,inducinganaveragefoldincreasein
expressionof9.0±1.4forPrnpSAM1and10.56±0.65forPrnpSAM2,butnoincreasewas
observedifthesgRNAwasomitted(0.97±0.07,Fig5A).IncreasedlevelsofPrPweredetected
onimmunoblotsindicatingtheextramRNAswerefunctional(Fig5B).
StimulationofPrnpwithCC9SAMsleadstooverexpressionof
extracellularmembrane-anchoredPrP
PrPcfunctionsinhealthanddiseasedependonitscorrectlocalizationinthemembrane,
attachedtotheoutsidesurfacebyaGPI-anchor[7,58].Wethereforeperformedextracellular
antibodylabelingexperiments,followedbyFACSandimmunocytochemistry(ICC)analyses,
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