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1 Morrisey(Lab(Protocol:(( Generating(Large((>1kb)(Genomic(Deletions(Using(CRISPRs( bydanswarr&davefrank GeneralComments genomicdeletions(>1kb),inordertoremoveorinactivateagenomicelementof promoter,tss,andfirstexon)byusingtwoguidernastomakemakingtwocuts, justupstreamanddownstream,oftheelementofinterest. Ifyouareinterested in considerusingthemorestandardapproachofusingasingleguidernatargetedto thefirstportionofthecodingregiontointroduceaframeshiftmutation.asonlyone guidernaisrequired,lesscloningandscreeningisnecessaryandefficiencymaybe better.however,thisapproachofcoursenotsuitableforstudyingthefunctionof GeneralPrinciples Generatingdeletionsofseveralhundredkbhasbeenreportedintheliterature, althoughcanver,etal(2014)demonstratedthattheefficiencyofgenomeeditingis roughlyproportionaltothesizeofthedeletionbeingintroduced. facilitateseaseofpcrgenotyping,butgeneratinglargerdeletionsarecertainly possible. Bycutting5 tothegenomicelementofinterestwithonesgrna,and3 tothe genomicelementofinterestwithasecondsgrna,theinterveningsegmentofdna fractionofcells. PCRprimersflankingthisregion(seeFigure1)canbeusedtoeasilygenotypecells usingstandardpcr. Figure1

2 DesigningtheCRISPRGuideRNAs 1. Choose250bpofsequence5 totheregionofinterest,and250bpofsequence3 to theregionofinterest. 2. GuideRNAscanbedesignedusingtheZhenglabdesigntoolathttp://crispr.mit.edu 3. Enterthefollowinginfo: a. Nameforsequence b. c. Sequencetype d. Species 4. Firstpasteyour5 sequenceintothebrowserandsubmitthejob. 5. Whileitisloading,submitasecondjobwithyour3 sequence. 6. Onceyourjobiscomplete,besuretosaveallofthedatafiles,includinglistsofall 7. Choosethetopthreetargetsequencesonthe5 end,andthetopthreeforthe3 end. Designingoligostoorder(SeeFigure2) 1. TheMITCRISPRdesigntoolwillinputa21bptargetsequence,followedbya3bp PAMsequenceingreenletters.DO(NOTincludethePAMsequencewhenordering youroligos. 2. Fortheforwardoligo,simplyadd tothe5 endofthetargetsequence. 3. Togeneratethereverseoligo: a. Takethereversecomplementofthetargetsequence b. Add tothe5 tothe3 end. 4. Orderthesestandardoligosforall6targets(threepairsforthe5 end,threepairs forthe3 end)fromidt 25nmolismorethansufficient Figure2 DesigningGenotypingPrimers and3 cutsites, respectively. present.

3 easilywithstandardpcrconditions.forlargerdeletions,youmayneedto alternativegenotypingstrategy. Phosphorylatingandannealingtheoligos 1. Setupthefollowingreactionforeachpairofoligos: - 2 ul forward oligo (100µM) - 2 ul reverse oligo (100µM) - 2 ul 10X T4 DNA Ligase Buffer (NEB) - 13 ul ddh2o - 1 ul T4 PNK (NEB) - 20 ul total 2. Anneal in a thermocycler using the following parameters: - 37 o C 30 min - 95 o C x 5 min, then ramp down to 25C at 5 o C/min - 4 o C hold Cut and Dephosphorylate the PX330/PX459 Vector: 1. Digest 1-2ug of PX330 or PX459 with BbsI for 1hr at 37 o C. 2. Heat-inactivate the RE at 65C x 15 minutes. 3. Add 1uL of calf intestinal phosphatase (CIP) to the reaction mixture, and incubate for 37C x 1hr. 4. Gel purify digested vector using QIAquick Gel Extraction Kit. Ligate Oligos into the PX330/PX459 Vector: 1. Set up the following ligation reactions: - 150ng of digested/dephosphorylated PX330/PX459-1uL phosphorylated/annealed oligo duplex - 4uL 5X T4 DNA ligase buffer (Invitrogen) - 1uL T4 DNA Ligase - ddh20 to bring reaction volume to 20uL 2. Incubate ligation reaction at 37C x 1hr or 16C overnight Transformation of Stbl3 Cells 1. Thaw Stbl3 cells on ice 2. Add 2uL of ligation reaction to 20uL of Stbl3 cells 3. Let sit on ice for 30 minutes 4. Heat shock at 42C for 45 s 5. Recover on ice for 2 minutes 6. Add 150uL SOC media 7. Incubate at 37C with shaking for 1 hour 8. Plate entire reaction onto LP/amp plates Screening Clones 1. After picking colonies and mini-prepping DNA, you can screen for inserts by either

4 performing double-digest with BbsI & AgeI (single band = oligo successfully annealed into vector; 2 bands indicates vector only without insert) followed by confirmatory sequencing, or proceeding directly to sequencing 2. Sequence positive clones using the human U6 primer: 5 ACT ATC ATA TGC TTA CCG TAA C 3 Screen for Cutting Efficiency **Werecommendscreeningall9combinationsofguideRNAsforcuttingefficiencyinacell linethatiseasytoworkwithandtransfect,suchas3t3s(mouse)or293ts(human)before proceedingwithdownstreamexperiments.screeningcanbeperformedwithmini@prepped DNA.** 1. Plate3T3or293Tcellsinto10wellsofeither6@wellor12@wellplatesinantibiotic@ freemedia,atadensityof50@70%confluency.thiswillallowforthe9combos+ GFPonlytransfectioncontrol(weusepMaxGFP+). 2. Transfecteachwellwith1ugofeach5 and3 construct(total2ugdna)fora12@ wellplate,or2.5ugofeach5 &3 construct(total5ugdna)fora6wellplateusing lipofectamine. 3. Forexample,thefollowingtransfectionprotocolmaybeusedfor6@wellplates: a. Combine2.5ugofeach5 and3 constructinasingletube,dilutedtoafinal volumeof150ulwithopti@mem. b. PrepareaLipofectaminemastermixinOPTI@MEMbyadding110uL lipofectamineto1540ulopti@mem(1xreactionmix:10ullipofectamine+ 140uLOPTI@MEM).Thiswillprovideadequatereagentsforall9testpairs, plusanadditionalgfpcontrol.incubateatroomtempx5minutes. c. Add150uLofthelipofectaminemastermixtoeachDNAmix(totalvolume 300uL).Incubateatroomtempx20@30minutes. d. Addall300uLoflipofectamine:DNAmixdropwisetocells 4. Transfectcellsinthelateafternoonorevening,andchangemediathefollowing morning hoursafterselection,addpuromycinifdesired(2ug/mLmediaworkswellfor 3T3cells)ifyouareusingthePX459backbone. 6. Leavecellsundisturbedfor36@48hours. Genomic DNA Extraction & PCR Genotyping 1. Roughly 3 days after transfection, remove media from cells, place plate on ice and add 300uL of 5-Prime Cell Lysis solution. 2. Scrape cells with cell scraper, pipette cells/cell lysis solution up/down several times with 1000uL pipette, and transfer to clean eppendorf tube. 3. Vortex x 10 seconds to lyse cells. If cell clumps are still visible, incubate at 37C until suspension is homogenous. 4. Add 1.5uL RNaseA to sample, invert several times and then incubate at 37C for 5 minutes. 5. Cool sample to room temperature by placing on ice for 1 minute 6. Add 100uL of 5-Prime Protein Precipitation solution 7. Centrifuge at max rate for 5 minutes

5 8. Transfer supernatant to clean eppendorf tube, then add 300uL isopropanol to precipitate DNA 9. Invert several times, then centrifuge at max rate x 5 minutes. 10. Wash DNA pellet with 70% ETOH, air dry, and then re-suspend in uL (goal concentration 200ng/uL). 11. Perform genotype using standard PCR. If your CRISPRs work, you should see a strong mutant band. The relative intensities of the wild-type and mutant band will give you a general sense of how efficient your CRISPR pairs are. 12. Pick your best pair based on their relative efficiency, as well as the best off-target score (from the MIT design tool). Maxi-prep these two plasmids for future experiments. GenerationofMonoclonalCellLinesusingLimitingDilution* 1. Usingyourcelllineofinterest,transfectasingle6@wellusingtheprotocoldescribed above(or,transfectanequivalentnumberofcellsusingthetransfectionprotocol appropriatetoyourcelllineofinterest) 2. Changetofreshmediathemorningaftertransfection.Ifyouplantousepuromycin selection,addanappropriatedose24hoursaftertransfection. 3. Youmayproceedtoisolationofsingleclonesasearlyas60@72hoursafter transfection.however,somecelllinesmaynottoleratethestressofpuromycin selectioncombinedwiththestressoflimitingdilution.considerchangingtofresh media(withoutpuromycin)after48hoursofpuromycinselection,andallowingthe cellstorecoverfor2@3daysbeforeproceedingtolimitingdilution. 4. Toperformlimitingdilution: a. Trypsinizethetransfectedcells,spindownandresuspendinfreshmedia b. Measurecelldensitywithahemocytometer c. Dilutecellstoafinalconcentrationof1cellper200uL(5cells/mL) d. Youwillneedtodothisusingaserialdilutionapproach,transferringatleast 1mLmediaperdilutioninordertoreducevariability.Forexample,ifyou determineyourcellsareataconcentrationof125,000cells/ml,dilutethem asfollows: i. Add1mLcellsuspensionto9mLfreshmediatoproduceDilution1at aconcentrationof12,500cells/ml. ii. Add1mLofDilution1to9mLfreshmediatoproduceDilution2,ata concentrationof1,250cells/ml. iii. Add1mLofDilution2to9mLfreshmediatoproduceDilution3,ata concentrationof125cells/ml. iv. Add1mLofDilution3to24mLoffreshmedia. e. Usingthefinaldilutionat5cells/mL,pipette200uLintoeachwellofa96@ wellplateusingamulti@channelpipette.plateout1@296wellplates. 5. Leaveplatesundisturbedforatleast3days.Changetofreshmediaafter3@5days. Notethatitwillbeverydifficulttoseecells/coloniesforatleast3@5days,anditwill likelytakeatleastaweekbeforecoloniesarelargeenoughtobesplitontoalarger plate. 6. Oncecoloniesareclearlyvisible(besuretoeliminatewellsthathavetwoormore distinctcolonies),washeachwellwithpbs,thentrypsinizecellswith50ultrypsin.

6 7. Add50uLofcelltrypsinsuspensionto1mLfreshmediaina24wellplate.Allow cellstogrowuntiltheyhavereachedanappropriatedensity. 8. suspensionintotwowellsofa12wellplate(perclone)withasmuchvolumeasthe nd wellwillbeforfurther expansionofthecellline). 9. genotypingpcr. 10. Expandthedesiredcelllinesasappropriate,andproceedtodownstream experiments *Notethatnotallcelltypeswilltoleratelimitingdilution,asitisquitestressfulformany celltypestobecompletelyisolatedfromothercells.youshouldstronglyconsidertrying beforeattemptingyourcrisprdeletions.alsonotethatifyourdeletionsignificantly affectscellproliferationorviability,youmaynotbeabletosuccessfullyisolatecellswith thedeletionofinterestusingthisprocedure. GenerationofKOMice 1. and3 constructusinggenecleanturbocolumns,perthe manufacturer sprotocol(followingtheinstructionforpurificationofpcrproducts) 2. ethanol. 3. Tris/0.1mMEDTA,pH7.5preparedwithdistilledwater) 4. PassthepurifiedDNAthroughaMilliporepurificationspincolumn %ethanol SubmitsamplestothePennTransgeniccoretobeinjectedtogetherina1:1ratio (usingeitheradoseof2.5ngor5ngperconstruct,totaldnadose5ngor10ng) 8. GenotypeF0generationusinggenotypingprimersasdescribedabove References 1. CongL,etal.Science Feb 15;339(6121): Fujii W, et al. Nucleic Acids Res Nov 1;41(20):e Ran FA, et al. Nat Protoc Nov;8(11): Canver MC, et al. J Biol Chem Jun 6.