Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse

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Method

Article

Two

CRISPR/Cas9-mediated

methods

for

targeting

complex

insertions,

deletions,

or

replacements

in

mouse

Kyriel

M. Pineault

a,b

,

Ana

Novoa

b

,

Anastasiia

Lozovska

b

,

Deneen

M. Wellik

a,1

,

Moises

Mallo

b,

*

,1

a

DepartmentofCellandRegenerativeBiology,UniversityofWisconsin-Madison,USA

b_Instituto_Gulbenkian_de_Ciência,_Portugal

ABSTRACT

Geneticallymodiﬁedmodelorganismsarevaluabletoolsforprobinggenefunction,dissectingcomplexsignaling

networks,studying human disease,and more.CRISPR/Cas9 technologyhas signiﬁcantlydemocratizedand

reducedthetimeandcostofgeneratinggeneticallymodiﬁedmodelstothepointthatsmallgeneeditsarenow

routinely and efﬁciently generated in as little as two months. However, generation of larger and more

sophisticatedgene-modiﬁcationscontinuestobeinefﬁcient.AlternativewaystoprovidethereplacementDNA

sequence,methodofCas9delivery,andtetheringthetemplatesequencetoCas9ortheguideRNA(gRNA)haveall

beentestedinanefforttomaximizehomology-directedrepairforprecisemodiﬁcationofthegenome.We

presenttwoCRISPR/Cas9methodsthathavebeenusedtosuccessfullygeneratelargeandcomplexgene-editsin

mouse.Intheﬁrstmethod,theCas9enzymeisusedinconjunctionwithtwosgRNAsandalongsingle-stranded

DNA(lssDNA)templatepreparedbyanalternativeprotocol.Thesecondmethodutilizesatetheringapproachto

coupleabiotinylated,double-strandedDNA(dsDNA)templatetoaCas9-streptavidinfusionprotein.

Alternativemethodforgeneratinglong,single-strandedDNAtemplatesforCRISPR/Cas9editing.

DemonstrationthatusingtwosgRNAswithCas9-streptavidin/biotinylated-dsDNAisfeasibleforlargeDNA

modiﬁcations.

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ARTICLE INFO

Methodname:CRISPR/Cas9-mediatedlargegeneticmodiﬁcationsinmouse

Keywords:CRISPR/Cas9,Gene-editing,Mouse,Zygote,Microinjection,ssDNA,Streptavidin/biotin

Articlehistory:Received23July2019;Accepted5September2019;Availableonline10September2019

*Correspondingauthor.

E-mailaddresses:kpineault@wisc.edu(K.M. Pineault),anovoa@igc.gulbenkian.pt(A.Novoa),alozovska@igc.gulbenkian.pt

(A.Lozovska),wellik@wisc.edu(D.M. Wellik),mallo@igc.gulbenkian.pt(M.Mallo).

1

Co-seniorauthors.

https://doi.org/10.1016/j.mex.2019.09.003

ContentslistsavailableatScienceDirect

MethodsX

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Methoddetails Animaluse

AllanimalexperimentswereperformedfollowingPortuguese(Portaria1005/95)andEuropean (Directive2010/63/EU)legislationsregardinghousing,husbandry,andwelfare.Thisprojectreceived reviewandapprovalbytheEthicscommitteeofInstitutoGulbenkiandeCiênciaandbythePortuguese NationalEntity,DirecçãoGeraldeAlimentaçãoVeterinária(licensereference:014308).

Commonmaterialsforallprocedures DNase/RNase-freewater

RNase-freeEppendorftubes RNase-freeﬁlteredpipettetips Thermocyclersystem

Heatblockorwaterbath Agarose

1xTAEbuffer

Gelelectrophoresissetup(chamberandpowersupply) Gelloadingdye

DNAladder(100bpor1kbladder)

DNAstain(ethidiumbromideoralternative) Gelimagingsystem[ultraviolet(UV)imaging] Temperaturecontrolledcentrifuge

NanoDropspectrophotometerorsimilarequipment

NOTE1:Commonmaterialsandequipmentcanbeusedfromanyreliablecompany. NOTE2:AllprotocolsshouldbeperformedunderDNase/RNase-freeconditions. A.invitrotranscriptionofCas9orCas9-streptavidinmRNA

Materials.

Cas9orCas9-streptavidinplasmid(thisprotocolisintendedforplasmidscontainingaT7promoter) Cas9:pT7-Cas9(availablecommerciallyfromOriGeneCat.No.GE100014)

Cas9-streptavidin:PCS2+Cas9-mSA(availablefromAddgenePlasmidNo.103882)

NOTE: TheSP6 promoterin thisplasmidwas replaced witha T7 promoterusing standard cloningtechniques.

NOTE:ProtocolcanbeadaptedtouseaplasmidcontaininganSP6promoter. RestrictionenzymeappropriatetolinearizeCas9plasmid(seenote1c) Phenol/Chloroform

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100%ethanol

mMessagemMachineT7Ultrakit(ThermoFisherCat.No.AM1345)

NOTE:UseSP6invitrotranscriptionkitifusingaplasmidcontaininganSP6promoter. RNeasyMiniKit(QiagenCat.No.74104)

Dryice Protocol

RestrictiondigestandcleaningofCas9orCas9-streptavidinplasmid.

1 PerformarestrictiondigestoftheCas9orCas9-streptavidinplasmid.Incubatereactionovernight atappropriatetemperatureforrestrictionenzyme.

a Basicreactionconditions:Plasmid10

m

g10xEnzymeBuffer5

m

lRestrictionEnzyme2

m

lWaterto 50

m

l

bNOTE:Obtainalargeamountofplasmidthroughstandardmidi-prepormaxi-prepprocedures. cNOTE:RestrictionenzymeshouldlinearizetheplasmidimmediatelyaftertheCas9or Cas9-streptavidinsequencewithintheplasmid.Thisallowsfor“runoff”transcription.Itispossibleto useanenzymethatcutsmultipletimeswithintheplasmidbackboneaslongasitgeneratesa linearpieceofplasmidcontainingtheT7primingsiteandtheentireCas9orCas9-streptavidin sequence.

2 Runasmallamount(₂₀₀ng)ofthecleanedrestrictiondigestona0.8%agarosegeltocon_ﬁrm plasmidwasproperlylinearized.

3 Add50

m

lofDNase/RNase-freewatertobringthereactionto100

m

l.

4 Add100

m

lofphenol/chloroformandshakevigorouslyuntilreactionbecomesopaque. 5 Spinsolutionatmaximumspeedfor5minatroomtemperature.

6 Carefullyremovetheupper,aqueouslayerofthereactionandmovetoanewtube. dNOTE:Itisveryimportanttoavoidcontaminationwiththelowerlayer.

7 Add1/10thvolume(10

m

l)of3MNaOAc,pH5.3,andmixwell. 8 Add2.5volumes(250

m

l)oficecold100%ethanolandmixwell. 9 Chillondryicefor>30minorat-20CovernighttoprecipitateDNA. 10Spinreactionatmaximumspeedfor30minat4_C.

11 CarefullyremovesupernatanttakingcarenottodisturbtheDNApellet. 12WashDNAbyadding500

m

lofice-cold70%ethanol.

13Spinatmaximumspeedfor10minat4_C.

14CarefullyremoveallofthesupernatanttakingcarenottodisturbtheDNApellet.

15Air dry DNA pellet at room temperature for 5–10minutes or until all traces of liquid have evaporated.

16ResuspendDNAinasmallvolume(15

m

l)ofDNase/RNase-freewaterandmeasureconcentration onaNanoDroporsimilar.

eNOTE:ThedesiredDNAconcentrationisbetween1–2

m

g/

m

ltohaveenoughDNAtemplatefor thesubsequenttranscriptionreaction.

Cas9 or Cas9-streptavidin mRNA synthesis: mMESSAGE mMACHINE T7 Ultra Kit. Follow the manufacturer’sprotocolwiththefollowingnotes:

1 Incubatethetranscriptionreactionfor2hat37_C.

2 Increaseto2

m

lTURBODNase,mixwell,andincubatefor15minat37C. aPropermixingwillensurethatalltracesofDNAtemplateareremoved. 3 Save2

m

lofthesolution(Sample1)beforeaddingtheE-PAPreagent. 4 IncubatewiththePoly(A)tailingreagentsfor30minat37C.

5 Save2

m

lofthesolution(Sample2)aftercompletingthePoly(A)tailingprocedure. a Dilutebothreservedsampleswith9

m

lofwaterandaddRNase-freegelloadingdye. bRunsamplesona0.8%agarosegeltodetermineifPoly(A)tailingwassuccessful(Fig.1).

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CleanCas9orCas9-streptavidinmRNA:RNeasyMiniKit. Followthemanufacturer’sprotocolwiththe followingnotes:

1Elutein40

m

lofRNase-freewaterinsteadofprovidedelutionbuffer. 2MeasuretheconcentrationoftheelutedRNAonaNanoDropofsimilar.

3Run200–500ngofmRNAona0.8%agarosegel(Sample3)toconﬁrmthatithasnotdegraded duringthecleaningprocedure(Fig.1).

4StoremRNAat-80C.

a NOTE: Aliquotintosmallervolumesand avoid freeze-thawcycles which might degradethe product.RunasmallamountofmRNAonagelpriortoeveryexperimenttoinsureproducthasnot degraded.

B.invitrotranscriptionofsgRNA(skipthisprocedureifpurchasingcommerciallysynthesizedsgRNA) RefertomethodvalidationforfurtherinformationonchoosingsgRNA(s)

Materials

sgRNAplasmid(protocolisintendedforplasmidscontainingaT7promoterduetorestrictionsof MEGAshortscriptT7kit).

sgRNA plasmids can be made in-house (e.g. Using pT7-sgRNA commercially available from OriGeneCat.No.GE100025)orpurchasedfromnumerousvendors.

LinearT7-sgRNAtemplate canalsobegenerated viaPCR. Proceedimmediatelywithin vitro transcriptionifusingthismethod.

RestrictionenzymeappropriatetolinearizesgRNAplasmid(seenote1c). Phenol/Chloroform.

3MNaOAcpH5.3. 100%ethanol

Fig.1.SynthesisofCas9orCas9-streptavidinmRNA.ImagesofGelSafestained0.8%agarosegelsofsamples1–3ofpreparation ofCas9-streptavidinmRNA(sectionA).Leftgelshowsexpectedsizeincreasepre-andpost-Poly(A)tailing(samples1and2)and asinglenon-degradedCas9-streptavidinmRNAproductaftercleaning(sample3).RightgelshowsexampleofappropriatePoly (A)tailing(samples1and2)butdegradationoftheCas9mRNAproductfollowingcleaning(sample3).Ladder:1kbladder,1kb and3kbbandsmarked.

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MEGAshortscriptT7Kit(ThermoFisherCat.No.AM1354)

MEGAcleartranscriptionclean-upkit(ThermoFisherCat.No.AM1908) Dryice

Protocol

RestrictiondigestandcleaningofsgRNAplasmid.

1 Perform a standard restriction digest of the sgRNA plasmid. Incubate reaction overnight at appropriatetemperaturefortherestrictionenzyme.

a Basicreactionconditions:Plasmid25

m

g10xEnzymeBuffer5

m

lRestrictionEnzyme2-3

m

lWaterto 50

m

l

bNOTE:Obtainalargeamountofplasmidthroughstandardmidi-prepormaxi-prepprocedures. cNOTE:RestrictionenzymeshouldlinearizetheplasmidimmediatelyafterthesgRNAscaffold withintheplasmid.Thisallowsfor“runoff”transcription.Itispossibletouseanenzymethat cutsmultipletimeswithintheplasmidbackboneaslongasitgeneratesalinearpieceofplasmid containingtheT7primingsiteandtheentiresgRNAscaffold.

2 Runasmallamount(200ng)ofthecleanedrestrictiondigestona0.8%agarosegeltoconﬁrm plasmidwasproperlylinearized.

3 Add50

m

lofDNase/RNase-freewatertobringthereactionto100

m

l.

4 Add100

m

lofphenol/chloroformandshakevigorouslyuntilreactionbecomesopaque. 5 Spinsolutionatmaximumspeedfor5minatroomtemperature.

6 Carefullyremovetheupper,aqueouslayerofthereactionandmovetoanewtube. aNOTE:itisveryimportanttoavoidcontaminationwiththelowerlayer.

7 Add1/10th_volume₍₁₀

_m

_l)_of₃_M_NaOAc_pH_5.3_and_mix_well. 8 Add2.5volumes(250

m

l)oficecold100%ethanolandmixwell. 9 Chillondryicefor>30minorat-20CovernighttoprecipitateDNA. 10Spinreactionatmaximumspeedfor30minat4C.

11 CarefullyremovesupernatanttakingcarenottodisturbtheDNApellet. 12WashDNAbyadding500

m

lofice-cold70%ethanol.

13Spinatmaximumspeedfor10minat4_C.

14CarefullyremoveallofthesupernatanttakingcarenottodisturbtheDNApellet.

15Air dry DNA pellet at room temperature for 5–10minutes or until all traces of liquid have evaporated.

16Resuspend DNA in a small volume (15

m

l) of DNase/RNase-free water and measure the concentrationonaNanoDroporsimilar.

aNOTE:ThedesiredDNAconcentrationisbetween1–2

m

g/

m

ltohaveenoughtemplateDNAfor thesubsequenttranscriptionreaction.

InvitrotranscriptionofsgRNA:MEGAshortscriptT7kit. Followthemanufacturer’sprotocolwiththe followingnotes

1 Incubatethetranscriptionreactionat37_C_for₄_h.

aIfdrops accumulatein thetube’slid,collectthereactioncontentswithashortspinatroom temperature(advisable,every1.5h).

2 Increaseto2

m

lTURBODNase,mixwell,andincubatefor30minat37_C. aPropermixingwillensurethatalltracesofDNAtemplateareremoved.

CleaningsgRNAtranscript:MEGAcleartranscriptionclean-upkit. Followthemanufacturer’sprotocol withthefollowingnotes

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1MakesureallcentrifugationstepsdonotexceedanRCFof15,000xg.Thiswillpreventdamagetothe spincolumns.

2UseRNAelutionoption1.Elutein50

m

lofDNase/RNase-freewaterandnottheprovidedelution buffer.Incubateat70_C_in_a_heat_block_for₁₀_min.

3Donotrepeattheelutionprocedureassuggestedbythemanufacturer.Inourhandsthisdoesnot increasetheRNAyieldssigniﬁcantlyandreducesﬁnalsgRNAconcentration.

4Followingcleaning,measuretheRNAconcentrationonNanoDroporsimilar.

5Run500ngona2%agarosegeltoconﬁrmproductionofasinglesgRNAproduct(Fig.2). a NOTE:InsomecasestheRNAmightgivetwoapparentproducts,onewiththeexpectedsizeand

anotherabitbigger.ThispatternresultsfromtheRNAacquiringdifferentconformationsthatrun differentlyinthegel.

6StoresgRNAat-80C

a NOTE:Aliquotintosmallervolumesandavoidfreeze-thawcycleswhichmightdegradetheproduct. RunasmallamountofsgRNAonagelpriortoeveryexperimenttoinsureproducthasnotdegraded.

C.SynthesisofsinglestrandedDNA(ssDNA)template Materials.

Targetingplasmidtemplate

Refertomethodvalidationforfurtherinformationondesignoftargetingplasmidtemplate. Primersetfortemplategeneration(forwardandreverse)

Oneprimermustcontainabiotinmodiﬁcationonthe50end

HighFidelityTaqPolymerasekitwithproofreadingactivity(e.g.HotStarHiFidelityPolymerasekit, QiagenCat.No.202602)

PCRpuriﬁcationsystem(e.g.QIAquickPCRpuriﬁcationkit,QiagenCat.No.28104) DynabeadsMyOneStreptavidinC1beads(ThermoFisherCat.No.65001)

Magnetictuberack 3MNaOAc,pH5.3 1MTrisHCl,pH7.5 100%ethanol BufferA 10mMTrisHClpH7.5 30mMNaCl 1mMEDTA BufferB

Fig.2. SynthesisofsgRNA.ImageofGelSafestained2%agarosegelofﬁnalsgRNAproduct(sectionB).Singlebandisobserved showingnosignsofdegradationfollowingprocedure.Ladder:1kbladder,1kband3kbbandsmarked.

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0.15 1mMEDTA Dryice

Tris-EDTA(TE)buffer 10mMTrisHCl 1mMEDTA Protocol

1 PCRprimersshouldbedesignedtoamplifydesiredgene-editedsequenceandcontain100-200bp ofﬂankinghomologyupstreamanddownstreamtothesiteofeditingwithinthegenome. a NOTE:OnlyonePCRprimershouldcontainabiotinmodiﬁcationonthe50end.

bNOTE:Ithasnotbeenempiricallytestedwhetherthechoiceofprimercontainingthebiotin modiﬁcation(forwardorreverse)affectstheefﬁciencyofgene-editing.

2 Setup5–10PCRreactionstoamplifytemplatesequencefromthetargetingplasmidtemplate. a NOTE: Use of a proof-readingenzyme is essential toreduce theprobability of introducing

mutations.

bNOTE:Minimizetheprimerconcentrationasmuchaspossibletoallowformoreefﬁcientoligo removalduringstep3.

cNOTE:CheckPCRreactionbyrunningasmallamount(2–5

m

lofthereaction)onanagarosegel tocon_firmreactiongeneratessinglePCRproduct.Ifreactioncannotbeoptimizedtoeliminate un-desiredbands,proceedwithagelPCRpurificationmethodtoobtaindesiredband. 3 PoolPCRreactionsandcleanusingacolumn-basedPCRpurificationsystemtoremovetheunused

biotinylatedprimerthatwillinterferewiththereactioninstep4.Thetypicalelutionvolumeis 50

m

l.EluteinDNase/RNase-freewaterinsteadofprovidedelutionbuffer.

a Reserve2

m

lofcleanedproducttoserveasaloadingcontrol(Sample1).

bCalculatethetotalyieldofpuriﬁedDNA.Thiswillbenecessarytocalculatethevolumeofbeads necessaryinstep4.

4 Wash Dynabeads MyOne Streptavidin C1 beads twice with 500

m

l of Buffer A. Use 1.5mL Eppendorfftubeswithamagneticracktoprecipitatebeads.

aFollow manufacturer’s recommendations to determine volume of beads required to bind biotinylatedDNA.

5 DilutepuriﬁedPCRproductin300

m

lofBufferAandincubatewithstreptavidinbeadsatroom temperaturefor1hwithrotation.

aNOTE:ForPCRproductsgreaterthan1kb,increasetheincubationtimetoseveralhoursorto overnighttoincreaseefﬁciencyofbiotin/streptavidinbinding.

6 Collectbeadsusingamagneticrackandsavethesupernatant(Sample2).Thiswillbeusedto estimatetheefﬁciencyofbiotin/streptavidinbinding.

7 Washbeads2x500

m

lwithBufferAatroomtemperature.

8 DenaturetheDNAtoliberatetheun-biotinylatedDNAstrand with300

m

lofBufferBatroom temperaturefor25minwithrotation.

aNOTE: ForPCRproductsgreaterthan 1kb,increaseelutiontime toseveralhoursforhigher efﬁciency.

9 Collectthebeadsusingamagneticrack.Carefullyrecoverthesupernatantensuringnobeadsare carriedover(Sample3).

a NOTE:DoNOTusenon-stickyEppendorftubesasthiswillresultinlosingthessDNAinthenext steps.

bNeutralizereactionwith30

m

lof1MTrisHCl,pH7.5. 10PrecipitateDNAfromSample2andSample3.

a Add30

m

lof3MNaOAc,pH5.3and900

m

lof100%ethanolandmixthoroughly. bPlacetubesondryiceforatleast30min.

11 RecoverprecipitatedDNAsbycentrifugationatmaximumspeedfor30minat4C.

aRemovesupernatantandallowDNApellettoair-dryfor5–10minoruntilanyremainingliquid hasevaporated.

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b ResuspendSample2in5

m

lofTEbufferorwater.

cResuspendSample3(ssDNA)inasmallvolume(11–15

m

l)ofRNase-freewater.

d NOTE:PrecipitatedssDNAfrequentlyadherestothetubewallalongtheentireverticallength insteadofformingapellet.Toresuspend,usethepipette-tiptomovethedropletofwateralong thetube.Liquidwillformdropletsalongthetube.Recovertheliquidwithashortcentrifugation andrepeattheprocessuntildropletsnolongeradheretothetubewall,whichmeansthatthe DNAisdissolved.Recoverthewholevolumewithashortspin.

12MeasuretheconcentrationofSample2andSample3onaNanoDroporsimilar. 13RunSamples1–3ona1%agarosegel(Fig.3).

a Run1–2

m

lofSample3(ssDNA).

b Trytousecomparableamountsofallsamplestoimprovecomparison.

c NOTE:ssDNArunsdifferentfromPCRproductsandsignalcanbedifﬁculttodetectusingmost gelvisualizationmethods.

14ssDNAcanbestoredat-20_C_or_-80_C.

D.SynthesisofbiotinylateddoublestrandedDNA(dsDNA) Materials.

Targetingplasmidtemplate

Refertosupplementalinformationforfurthernotesondesignoftargetingplasmidtemplate Primersetfortemplategeneration(forwardandreverse)

Bothprimersmustcontainabiotinmodiﬁcationonthe50 end.

HighFidelityTaqPolymerasekitwithproof-readingactivity(e.g.HotStarHiFidelityPolymerasekit, QiagenCat.No.202602)

Fig.3. GenerationofssDNAproduct.ImageofGelSafestained1%agarosegelofsamples1–3ofgeneratinga738bpssDNA product(sectionC).15.5mgofbiotinylatedPCRproductwasboundtostreptavidin-conjugatedbeads(sample1).1.8mgof unboundDNAwasrecoveredinthesupernatantfollowingbinding(sample2).2mgofssDNAproductwasrecoveredintheﬁnal steps(sample3).TheweakssDNAbandandsmearwithinthelaneiswhatistypicallyobserved.Approximately350ngofDNA (dsDNAorssDNA)wasloadedperlane.Ladder:100bpladder,200bp,500bp,and1200bpbandsmarked.

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PCRpuriﬁcationsystem(e.g.QIAquickPCRpuriﬁcationkit,QiagenCat.No.28104) Protocol

1 PCRprimersshouldbedesignedtoamplifydesiredgene-editedsequenceandcontain100-200bpof ﬂankinghomologyupstreamanddownstreamtothesiteofeditingwithinthegenome. 2 Setup5–10PCRreactionstoamplifytemplatesequencefromthetargetingplasmidtemplate.

a NOTE: Use of a proof-reading enzyme is essential to reduce the probabilityof introducing mutations.

bNOTE:Minimizetheprimerconcentrationasmuchaspossibletoallowformoreefﬁcientoligo removalduringstep3.

cNOTE:CheckPCRreactionbyrunningasmallamount(2–5

m

lofthereaction)onanagarosegelto conﬁrmreactiongeneratessinglePCRproduct.Ifreactioncannotbeoptimizedtoeliminate un-desiredbands,proceedwithagelPCRpuriﬁcationmethodtoobtaindesiredband.

3 PoolPCRreactionsandcleanusingaPCRpuriﬁcationsystem.Thisstepisessentialtoremovethe unusedprimersthatwillinterferewiththebindingoftheDNAtotheCas9-mSAmoleculeupon injection(sectionE).Thetypicalelutionvolumeis50

m

l.EluteusingRNase-freewaterandnot suppliedelutionbuffer.

aMeasureconcentrationofcleanedPCRproductonNanoDroporsimilar.

4 Runasmallamount(200–500ng)ofcleanedPCRproductonageltoconﬁrmsizeandpurityof product.

5 BiotinylateddsDNAcanbestoredat-20Cor-80C.

E.ConditionsformicroinjectionofCRISPR/Cas9reagents Materials.

Cas9protein(optionforMethod1only)

Cas9orCas9-streptavidinmRNA(seeprocedureA) sgRNA(s)(seeprocedureB)

ssDNAtemplate(seeprocedureC,forMethod1only)

Biotinylated-dsDNAtemplate(seeprocedureD,forMethod2only) Microinjectionbuffer

8mMTrisHCl,pH7.5 0.2

Method1:Gene-editingusingCas9,gRNA(s),andssDNAtemplate

Cas9protein 100ng/ml –

Cas9mRNA – 37.5ng/ml

sgRNA(s) 20ng/ml(eachifusingmultiple)

ssDNA 20ng/ml

Method2:Gene-editingusingCas9-streptavidinfusionproteinmRNA,gRNA(s),and Biotinylated-dsDNAtemplate

Cas9-streptavidinmRNA 35-40ng/ml

sgRNA(s) 20ng/ml(eachifusingmultiple)

Biotinylated-dsDNA 20ng/ml

CRISPR/Cas9 reagents were injected into the pronuclei of fertilized oocytes using standard procedures[1].Gene-editingcanbeassessedbyPCRanalysisand/orSouthernBlottingasappropriate forthedesiredmodiﬁcation.

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Fig.4. Generationoflargegeneticmodificationsinmouse.(A)InsertionoftwoLoxPsites700bpapartusingMethod1(section E).DiagramrepresentstargetingstrategyusingtwosgRNAs(purplearrows)andalssDNAtemplatetoinserttwoLoxPsites (orangetriangle)flankinganexon(bluebox).Homologyregionsarenotedbyblackdashedlines.RepresentativelocationsofPCR primers(blackarrows)withintheendogenousandeditedsequencearemarkedandexpectedbandsizesarelistedabovegel images.Gels(right)showPCRanalysisforthe50and30LoxPsitesof4founder(F0)animals,twoheterozygoteanimalsmarkedby blackarrows.(B)GeneswapofcodingexonsusingMethod2(sectionE).Diagramrepresentstargetingstrategyusingtwo sgRNAs(purplearrows)andabiotinylateddsDNAtemplatetoreplacetwoexons(blueboxes–endogenousexons,redboxes– swapexons)whilepreservingflankingandintronicsequence(blueline).Homologyregionsarenotedbyblackdashedlines.

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NOTE: It isofcritical importancetoprepare allstockreagents(Cas9/Cas9-streptavidinmRNA, sgRNAs,and replacementDNA) asconcentrated aspossible.Thus, whenreagents aredilutedfor microinjection, potential contaminants are diluted beyond detection and will not obstruct the injectionneedle.

Methodvalidation

WehaveusedbothMethod1andMethod2togenerateadiversearrayofgene-editedmouse modelsincluding;

ConditionalalleleviasimultaneousincorporationoftwoLoxPsites

GeneswapsthroughremovingandreplacingthecodingregionsofGeneAwithGeneB

For ourgeneediting strategies,potentialsgRNAs aremanuallyidentifiedtogenerateadouble strandbreakatthe50and30boundariesoftheregionofDNAtobeedited.ItisimportantthattheCas9 generatedcutsiteisascloseaspossibletothedesirededitingsite tomaximizetheefficiencyof homologydirectedrepair.Thus,therelativenumberofpotentialsgRNAstobeconsideredwillvary dependingonthenatureofthedesirededit.Publicallyavailableonlinetoolscanbeusedtoevaluate thepotentialefficiencyofsgRNAs;however,Cas9-mediatedcuttingefficiencyshouldbeempirically determinedforeachsgRNA.AfterthesgRNA(s)havebeenvalidated,thetargetingplasmidvectorcan be designed and generated using conventional cloning techniques or custom synthesized commercially.Weuse100-200bpofflankinghomologyforboththessDNAandbiotinylated-dsDNA targetingmethods.Thesizeofthehomologyarmisbasedonevidenceprovidedbyotherpublished CRISPRmethodologiesandexperienceinourlaboratory[2_–5].Atargetingplasmidcontaining300bp ormoreofflankinghomologyallowsenoughflexibilitytopickoptimalprimerpairsforgenerating DNAtemplates.

The choiceof using Method 1 or Method 2 must be determinedempirically and will differ dependingonthenatureofthedesirededit.Wehavesuccessfullygenerated“floxed”allelesusing Method1;however,noevidenceofeditingwasobservedwhenweattemptedtogenerateageneswap (Fig.4A).Alternatively,weachievedveryhighgeneeditingefficiencywhencreatingacomplexgene swapusingMethod2(Fig.4B).Itisimportanttonotethattheefficiencyofgeneeditingwillvary dependingonthetargetlocusandthenatureoftheeditbeinggenerated.Efficiencyofeditingalso dependsonthequalityoftheCRISPRreagentsusedformicroinjectionanditisofcriticalimportanceto insure that RNA based components show no signs of degradation prior to every round of microinjection.

Additionalinformation

ThecommercialavailabilityofCRISPR/Cas9reagentsandnumerousadvancementsinefficiencyof CRISPR-based gene editing have allowed this technology to be routinely used in laboratories throughoutthelifesciencesandmedicinedisciplines.Thegenerationofnovelgene-editedmouse modelsisnowsubstantiallyeasierandfasterthanusingconventionalstemcell-basedgene-editing technologies.CRISPR/Cas9isnowroutinelyusedforthegenerationofpointmutations,“indels”to createloss-of-functionalleles,andinsertionofDNAsequencesincludingproteintagslikeFLAGorHA andfluorescentreporterslikeGFP[6,7].However,efficienteditingand/orinsertionoflargesequences ofDNA,eveninthecaseoffluorescentreporters,remainabigchallengeinthefield.

RepresentativelocationsofPCRprimerstogenerateauniquebandifexonswapoccurs(Analysis1,greenarrowsandbrackets) areshowninupperdiagram,PCRprimerstoscreenforcompleteexonswap(Analysis2,bluearrowsandbrackets)areshownin lowerdiagram,expectedbandsizesarelistedabovegelimages.Gels(right)showPCRanalysisforexonswapsof16F0animals. BlackarrowsonAnalysis2gelsshowtwoanimalstargetedtohomozygousefﬁciency.Boxedregion,showninhigher magniﬁcationininset,demonstratesbandingpatternsforwildtype(WT),heterozygous(het),andhomozygous(homo) targetedExon2swap.Targetingplasmid(+)andwildtypegenomicDNA(WT)wereusedascontrols.

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Severalgroupshaveexploredmethodstoincreasetheefficiencyofhomologydirectedrepair(HDR) thoughtheactiverecruitmentortetheringofthetemplateDNAsequencetoeitherCas9orthesgRNA [11–14].TheCas9fusionproteintomonomericstreptavidinusedinMethod2describedherewas initiallydeveloped toimprovetheefficiencyof generatingfluorescent reporteralleles [15]. They report targeting efficiencies up to 97% when using a single sgRNA, Cas9-streptavidin, and a biotinylated-dsDNAtemplatetoknockinaparticularDNAsequenceintoaspecificpositioninthe genome.Themaindifferencebetweenthisreportandourmethodisthedemonstrationofeffective targetingusingtwosgRNAstoreplaceaparticulargenomicsequenceforanotherone(e.g.ageneor enhancerswap).Inourhands,theuseoftwoguidesdoesnotappeartointerferewithhomologous recombinationortheorientationoftheeditedsequence,asmightbepredicted.Thisnewmethod mightproveparticularlyusefulforgeneratingallelesrequiringgene-editingoflargesequencesof DNA.

DeclarationofCompetingInterest

Theauthorsdeclarenoconﬂictsofinterest Acknowledgements

WeacknowledgethehelpandservicesoftheAnimalFacilitystaffattheInstitutoGulbenkiande Ciência.ThisworkwassupportedbyPTDC/BEX-BID/0899/2014andLISBOA-01-0145-FEDER-030254 (FCT, Portugal) toM.M. and bythe NationalInstitute of Arthritis and Musculoskeletal and Skin DiseasesoftheNationalInstituteofHealth(NIH)AwardNumberR61AR073523-02toD.M.W. References

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