CasOT-a genome-wide Cas-gRNA off-target searching tool

BIOINFORMATICSAPPLICATIONSNOTEVol.30no.82014,pages1180–1182

doi:10.1093/bioinformatics/btt764

GenomeanalysisAdvanceAccesspublicationJanuary2,2014CasOT:agenome-wideCas9/gRNAoff-targetsearchingtoolAnXiao1,y,ZhenchaoCheng1,y,LeiKong2,ZuoyanZhu1,ShuoLin3,GeGao2,*andBoZhang1,*

1KeyLaboratoryofCellProliferationandDifferentiationoftheMinistryofEducation,2StateKeyLaboratoryofProteinandPlantGeneResearch,CenterforBioinformatics,CollegeofLifeSciences,PekingUniversity,Beijing100871,Chinaand3DepartmentofMolecular,CellandDevelopmentalBiology,UniversityofCalifornia,LosAngeles,CA90095,USAAssociateEditor:JohnHancock

ABSTRACTdifferentregionsoftheprotospacerinthetargetsitealsohaveSummary:TheCRISPR/CasorCas9/guideRNAsystemisanewlydifferenteffects:amismatchintheregionofthefirst12ntadja-developed,easilyengineeredandhighlyeffectivetoolforgenetarget-centtothePAM(calledtheseedregion)maycauseanotableing;ithasconsiderableoff-targeteffectsinculturedhumancellsandinreductionofthecleavageactivityofCas9/gRNA,whereasmis-severalorganisms.However,theCas9/guideRNAtargetsiteistoomatchesintheotherregionoftheprotospacer(thenon-seedshortforexistingalignmenttoolstoexhaustivelyandeffectivelyiden-region)haveamuchweakereffect(Congetal.,2013;Jiangtifypotentialoff-targetsites.CasOTisalocaltooldesignedtofindetal.,2013;Liuetal.,2013).

potentialoff-targetsitesinanygivengenomeoruser-providedse-Anapproachtoidentifypotentialoff-targetsitesinanygivenquence,withuser-specifiedtypesofprotospaceradjacentmotif,genomeorothersequenceisnecessarytoevaluatetheoff-targetandnumberofmismatchesallowedintheseedandnon-seedregions.effectsofCas9/gRNAinvivo.AnonlinetoolusedforCas9/Availability:http://www.77cn.com.cn/casot/gRNAtargetdesignalsosupportsthepredictionofcertainContact:zfgenetics@http://www.77cn.com.cnorbzhang@http://www.77cn.com.cnpotentialoff-targetsites(Hsuetal.,2013);however,neitheraSupplementaryInformation:SupplementarydataareavailableatdetailedscoringalgorithmnoranadjustableoptionisavailableBioinformaticsonline.inthistool,andonlyafewgenomescanbesearchedfortargetorReceivedonOctober7,2013;revisedonDecember6,2013;acceptedoff-targetsites.Otheronlinetools,TagScan(Iselietal.,2007)oronDecember24,2013PatternMatch(http://www.77cn.com.cn/medakavw/pattern-

match/),areavailabletosearchforregionsinsomecommonly

usedgenomesoronlyinthemedakagenome,withuptotwo

1INTRODUCTIONmismatches.Ontheotherhand,widelyusedheuristicalignmentCas9/gRNA(guideRNA),orCas9/singleguideRNA,acusto-toolslikeBLASTNandBLATarenotdesignedforexhaustivemizedtypeofclusteredregularlyinterspacedshortpalindromicsearchingforshortnucleotidesequences(asshortas 20nt).repeat/CRISPR-associated(CRISPR/Cas)system,hasrecentlyInaddition,thevariabletypesofPAMwithrandomnucleotidesbeenusedforeffectivegenetargetinginmanyorganismsandalsoneedtobeconsideredduringthesearchforpotentialoff-culturedcells(Pennisi,2013;Xiaoetal.,2013).Thissystemistargetsites.

easytodesignandengineer;onlyasingleshortgRNA,whichHere,wereportCasOT,adownloadableandopen-sourcetoolcontains 20-ntregionreversecomplementarytoonestrandofdesignedtoidentifypotentialoff-targetsitesofgivenCas9/thetargetDNA(theprotospacer)withan-NGGmotifin30-endgRNAtargetsortargetpairsinanyuser-specifiedsequenceor(theprotospaceradjacentmotif,PAM)ofthetargetsite,hastogenome,withseveraltunableparameterslikePAMtypeandthebesynthesizedforaparticulartargetsite.ThesameCas9nucle-mismatchnumberintheseed(upto4–6nt)andnon-seedregionsasecanbeuseduniversally;thisnotablyreducesthecostofboth(SupplementaryMaterialS1).

timeandlabor,promotingthebroadandrapidlydeveloping

applicationofthissystem(Jineketal.,2012).

However,thespecificityofCas9/gRNAneedscarefulevalu-2IMPLEMENTATIONANDFEATURESation.Forexample,Cas9/gRNAseemstohaveconsiderableoff-

targeteffectsinculturedhumancells(Cradicketal.,2013;Fu2.1Single-gRNAsearchingmode

etal.,2013;Hsuetal.,2013;Malietal.,2013a;Pattanayaketal.,CasOTisaPerlscriptandcanrunlocallyinWindows,*nixand2013)andinseveralorganisms(Fujiietal.,2013;Lietal.,2013;MacOSsystems.Threesearchingmodes,single-gRNA,paired-Shanetal.,2013).InvitroexperimentssuggestthatthePAMgRNAandtarget-and-off-target,areprovided.Inthesingle-typecouldinfluencethecleavageactivity:the-NGGtypeofgRNAmode,usersarerequiredtoprovide(i)aninputPAMshowsthehighestactivity,PAMof-NAGcomessecondFASTAfileincludingindividual21–33-nttargetsites(18–30-ntandPAMof-NNGGshowstheweakestactivity.MismatchesinprotospacerplusthePAM-NGGinthe30-end),(ii)genome

sequencefilestosearchforoff-targetsitesoranyuser-specified

*Tosequenceand(iii)(optional)agenomeannotationfile(Ensemblywhomcorrespondenceshouldbeaddressed.Theauthorswishittobeknownthat,intheiropinion,thefirsttwoGTFformat)fordeterminingwhetherthepotentialoff-targetauthorsshouldberegardedasjointFirstAuthors.sitesarelocatedinexons.Linkstopublicsequencesand1180ßTheAuthor2014.PublishedbyOxfordUniversityPress.Allrightsreserved.ForPermissions,pleasee-mail:journals.permissions@http://www.77cn.com.cnDownloaded from http://www.77cn.com.cn/ at Guangxi Univeristy on April 14, 2014

CasOT:aCas9/gRNAoff-targetsearchingtool

http://www.77cn.com.cnerscanspecifyseveralparametersforthesearch,includingtheallowedlevelofPAMtype(fourlevelsareavailable),andthemaximumnumberofmismatchesallowedintheseedregion(0–6areavailable,butallowing5or6mismatchesneedsalargecomputerrandomaccessmemory;twobydefault)andinthenon-seedregion(nolimitbydefault).

TheprogramreadstheinputtargetsequencesanddividesthemintoPAMs,seedregionsandnon-seedregions.Apoolisbuilttocontainallpossible12-ntsequencessimilartotheseedregionsandtheirreversecomplementarysequences,withmis-matchesnomorethanthatallowedbytheuser-specifiedoption.Thegenomesequencesorotheruser-providedsequencesarescannednucleotide-by-nucleotidetoidentifywhetherany12-ntfragmentappearsinthepre-builtseedpool.Iffound,ad-Fig.1.Sampleinputfileandpossibleoff-targettypesintheCasOTjacentgenomicsequencesofthefragmentareextractedandadd-pair …… 此处隐藏：9476字，全部文档内容请下载后查看。喜欢就下载吧 ……