reverse:5′-CAAGCAGAAGACGGCATACGAGATNNNNNNNwasdeterminedbytimeaftertransfectiondividedbythe
NGTCTCGTGGGCTCGG-3′.doublingtimeofHEK293Tcells(20h).
ThesequenceNNNNNNNNrepresentsindexes(16SMeta-
genomicSequencingLibraryPreparation.IlluminaTech.NoteIdentificationofnumbersofmutantalleles.Acustomscript
15044223Rev.A).PCRampliconswereindividuallypurified(availableatgithub:https://github.com/changxingihs/mutant-
usingAmpureBeads(BeckmanCoulter).Allthesampleswereallele-calculation-for-TAM/)incombinationwithSAMtools
pooledwithequalmolarratio.AmpliconsizedistributionwaswasusedtoidentifythemutantallelesinthesgRNA-targeted
determinedwithAgilentbioanalyzer,andampliconswerepurifiedsequences.Briefly,nucleotideswithsubstitutionfrequencies
usingAmpureBeads(BeckmanCoulter).Adetailedstep-by-step≥0.1%wereconsidered,andreadswiththesamesequencewere
procedureofcreatinggeneticvariantsbyTAMandsubsequentcombinedandcounted.Thenumbersofsubstitutionsofeach
42
analysisbyNGSisdescribedelsewhere.readswerealsocalculated.
Thefull-lengthsequencingadaptorsanddualindexesarebased
onNextra(Illumina).AllPCRswereconductedwithPhusionHigh-dCas9-AIDxfootprintanalysis.Todeterminethefootprint
FidelityDNApolymerase(NewEnglandBioLabs).Thesequenc-ofdCas9-AIDxonDNA,HEK293TcellswiththeeGFP-stop-
inglibrariesweresequencedonaMiseqforsingleend300bp,codonreporterweretransfectedwithindividualsgRNAstargeting
oronaHiseq2500forpairedend250bp.AAVS1(n=3),eGFP(n=4),ortheexon6ofABLkinasegene
Sequencingreadswerefirsttrimmedby20bponbothends(n=5).Sevendaysaftertransfection,genomicDNAwascollected
toremovePCRprimers.Onlyreads/basesabove(qualityandsgRNA-targetedregionsweresubjectedtohigh-througput
score)Q30wereretainedforsubsequentanalysis.Readsweresequencing.CtoTconversionsinawindowbetween?40bpand
mappedtotargetDNAusingBWA-MEM(arXiv:1303.3997v2+50bpfromthePAMsequencewerecalculated.
(q-bio.GN)).VariantswereidentifiedwithSAMtoolspile-up.
AllsequenceanalysiswasperformedrelativetotheplusPrimers.Thefollowingprimerswereusedtoamplifythetargeted
strandofDNA.genomicDNAorcDNA.Theuniqueprimersweresynthesized
Substitutionfrequencieswerecalculatedbyreadswithtoincludea33and34baseoverhangatthe5′endoftheforward
substitution/totalreadscoveringthatbase.Nucleotideswith>0.1%andreverseprimers,respectively(forward:5′-TCGTCGGCAGC
substitutionwereidentifiedasmutatedbases.Indelswereidenti-GTCAGATGTGTATAAGAGACAG-3′;reverse:5′-GTCTCGTG
43
fiedwithCRISPResso.GGCTCGGAGATGTGTATAAGAGACAG-3′).Primersarelisted
ToanalyzethesequencemotifsassociatedwithAIDx-orinSupplementaryTable1.
dCas9-AIDx-inducedmutations,nucleotides2bpupstream
and1bpdownstreamofthemutatedCbases(position0)
41.Wang,T.,Wei,J.J.,Sabatini,D.M.&Lander,E.S.Geneticscreensin
weredetermined.IfthemutatedbaseswereG,reversecom-
humancellsusingtheCRISPR-Cas9system.Science343,80–84(2014).
42.Chang,X.&Ma,Y.Q.UsingtargetedAID-mediatedmutagenesis(TAM)
plementsequenceswerederivedandcombinedwithsequences
todiversifyagenomiclocusinmammaliancells.Protoc.Exch.
withmutationsatC.Allsequencingdatawereanalyzedon
http://dx.doi.org/10.1038/protex.2016.062(2016).
GALAXYplatform,andplottedbyGraph-padPrism.To
43.Pinello,L.etal.AnalyzingCRISPRgenome-editingexperimentswith
determinetheaveragemutationrate,thenumberofcellcyclesCRISPResso.Nat.Biotechnol.34,695–697(2016).
nAturemethods
doi:10.1038/nmeth.4027 |
|
