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35320163RESEARCHANDEXPLORATIONINLABORATORYVol．35No．3Mar．2016CRISPR-dCas91，21．2000932．200092:CRISPR-Cas9，Cas9RuvCHNHdCas9，———。gRNA，RNA，，。CRISPR-dCas9、，。:CRISPR;dCas9;RNA;Q-7A1006－7167201603－0020－04TheStateoftheArtofCRISPR-dCas9SystemonRegulatingLevelofGeneExpressionLUOMin1GUHua21．ShanghaiKongjiangHighSchoolShanghai200093China2．SchoolofLifeScienceandTechnologyTongjiUniversityShanghai200092ChinaAbstractCRISPR-Cas9systemhasbeenwidelyconsideredasapromisinggenomeeditingtechnologyinmolecularbiologyresearch．TheengineereddCas9proteinbyinactivatingRuvCandHNHnucleasedomainsofCas9proteinplaysaroleingeneexpressionregulation．ResearchersmanipulatedthestructuresofgRNAthatcanattractRNAbindingproteinswhichfusetodifferenttranscriptionfactorsforthepurposeofactivatingorinhibitinggenetranscription．ThispaperreviewedthestructurefunctionmechanismandcurrentprogressofCRISPR-dCas9systemingeneexpressionregulation．KeywordsCRISPRdCas9gRNASAM2015－12－05814023991998－。E-mailluomin_kj@sina．com1974－。E-mailgu_hua@tongji．edu．cn0DNADNA“”DNADNA“”DNA。CRISPRclusteredregularlyinterspacedshortpalindromicrepeats-CasCRISPR-associated。CRISPRDNA550bpleader-repeat-spacerunits。24-48bp250。26～72bpspacer3，:CRISPR-dCas9CRISPRDNA。CRISPRCRISPRCRISPR-associatedCasCRISPRCRISPRleadersequence1。CRISPRCasCRISPRRNARNARNADNA。Cas、、1。CRISPR、。1CRISPR“n”2CRISPR-Cas9CRISPRIIRNARNAguideRNAgRNACas9。gRNAcrRNAtracrRNARNA。CRISPR-crRNACas9RNAIIIcrRNA。crRNACRISPRtracrRNARNACas9DNA3。gRNAcrRNAtracrRNA。Cas9gRNADNA。Cas9gRNAProtospacerAdjacentMotifPAM。PAMCas9。CRISPRIIPAMNGG3。Cas9、gRNAPAM。gRNACas9PAM3-4DoubleStrandBreakDSB4DNA。1CRISPR-dCas9CRISPR-Cas9。Cas9RuvCHNH。DNA。Cas9D10ACas9RuvCRuvC-HNHHNH+Cas9H840ACas9RuvCRuvC+HNHHNH-Cas9。RuvCHNHD10A＆H840ARuvC-＆HNH-Cas9dCas9deadCas95。dCas9DNAgRNADNA。dCas9DNAdCas9。dCas9gRNA2。VP64dCas96。dCas9VP64。VP64dCas93dCas910VP6414CRISPR-dCas9。Chavez16VP64-p65-RtadCas9VP64dCas9。KRABKox1dCas9CRISPR7。dCas9dCas94。gRNAdCas93。CRISPR-dCas9RNAlncRNAs。K5625lncRNAsH19MALAT1NEAT1TERCXISTCRISPR-dCas980%16。12352CRISPR-dCas9112CRISPR-dCas9Perez-Pinera4gRNA8ASCL1NANOGHBG1/2MYOD1VEGFATERTIL1BIL1R2。dCas9-VP644gRNAHEK293T84gRNA。RNADNADNARNA。TALENdCas9-VP64IL1RN8。dCas9。CRISPR-dCas9KonermannRNAsingle-guideRNAsgRNACRISPR-dCas9。sgRNARNA。sgRNARNAVP60RNA。synergisticactivationmediatorSAMdCas9121009。sgRNAsgRNA23000。PLX-4720。PLX-4720。sgRNA。SAM9。SAM。enhancer。RNAlongnon-codingRNAlncRNAlncRNADNA310。dCas9CRISPRRNACRISPR。CRISPRRNA11。CRISPR-dCas9sgRNAlncRNA。3lncRNA113CRISPR-Cas9。5Cas917。sgRNA。Hsu18sgRNA8-12bp。223，:CRISPR-dCas9RNAiRNAimRNAmRNADNADNARNAi。DNADNA-bindingdomainDBDs。DBDsZFsTALENs12。sgRNA、CRISPR-dCas9CRISPR-Cas9。RNAiDBDsCRISPR-dCas9、microRNAsRNAs。。tet-onMAVS293TdCas9/sgRNANF-κB、IRF3/7cJun/ATF2IFNB1CRISPR-dCas919。CRISPR-dCas9。93%20。CRISPR-dCas9RNA。References1PhilippeHRodolpheB．CRISPR/CastheImmuneSystemofBacteriaandArchaeaJ．Science20103275962167-170．2．CRISPR/CasJ．201551122063-2069．3SanderJDJoungJK．CRISPR-CassystemsforeditingregulatingandtargetinggenomesJ．NatureBiotechnology2014324347-355．4HsuPDLanderESZhangF．etal．DevelopmentandApplicationsofCRISPR-Cas9forGenomeEngineeringJ．Cell20141571262-1278．5ChengAWWangHYangH．etal．MultiplexedactivationofendogenousgenesbyCRISPR-onanRNA-guidedtranscriptionalactivatorsystemJ．Cellresearch201323101163-1171．6MaederMLLinderSJCascioVM．etal．CRISPRRNA-guidedactivationofendogenoushumangenesJ．NatureMethods20131010977-979．7GilbertLALarsonMHMorsutL．etal．CRISPR-MediatedModularRNA-GuidedRegulationofTranscriptioninEukaryotesJ．Cell20131542442-451．8PabloPPChristopherMV．etal．RNA-guidedgeneactivationbyCRISPR-Cas9-basedtranscriptionfactorsJ．NatureMethods20131010973-976．9KonermannSBrighamMDTrevinoAE．etal．Genome-scaletranscriptionalactivationbyanengineeredCRISPR-Cas9complexJ．Nature20155177536583-588．10TsaiMCManorOWanYetal．LongNoncodingRNAasModularScaffoldofHistoneModificationComplexesJ．Science20103295992689-693．11ZalatanJGLeeMEAlmeidaRetal．EngineeringComplexSyntheticTranscriptionalProgramswithCRISPRRNAScaffoldsJ．Cell20151601-2339-350．12MorbitzerRRomerPBochJetal．Regulationofselectedgenomelociusingdenovoengineeredtranscriptionactivator-likeeffectorTALE-typetranscriptionfactorsJ．ProcNatlAcadSciUSA．20101075021617-21622．13KhalilASLuTKBashorCJetal．AsyntheticbiologyframeworkforprogrammingeukaryotictranscriptionfunctionsJ．Cell20121503647-658．14ChakrabortySJiHKabadiAMetal．ACRISPR/Cas9-basedsystemforreprogrammingcelllineagespecificationJ．StemCellReports201436940-947．15ChavezAScheimanJVoraSetal．HighlyefficientCas9-mediatedtranscriptionalprogrammingJ．NatMethods2015124326-328．16GilbertLALarsonMHMorsutLetal．CRISPR-mediatedmodularRNA-guidedregulationoftranscriptionineukaryotesJ．Cell20131542442-451．17FuYFFodenJAKhayterCetal．High-frequencyoff-targetmutagenesisinducedbyCRISPR-CasnucleasesinhumancellsJ．NatBiotechnol2013319822-826．18HsuPDScottDAWeinsteinJAetal．DNAtargetingspecificityofRNA-guidedCas9nucleasesJ．NatBiotechnol2013319827-832．19．CRISPR/Cas9D．20151-133．20MauranoMTHumbertRRynesEetal．Systematiclocalizationofcommondisease-associatedvariationinregulatoryDNAJ．Science201233760991190-1195．檿