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CRISPR/Cas9*(100193)Clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)/CRISPR-associated(Cas)9(zincfingerendonucleaseZFN)(transcriptionactivator-likeeffectornucleaseTALEN)．、InDel、、．、．CRISPR/Cas、、．CRISPRCRISPR/CasQ7DOI:10.3724/SP.J.1206.2013.00215ProgressinBiochemistryandBiophysics2013,40(8):691~702*(CARS-37).**.Tel:010-62731142,E-mail:qingyong.meng@gmail.com2013-05-192013-07-29．．(engineeredendonucleaseEEN)．(zincfingerendonucleaseZFN)．DNAZFNFok[1]DNA．ZFN、、、、、、、、、、、、、iPS[2]．HIVZFN(CCR)[3]．ZFN．———(transcriptionactivator-likeeffectornucleaseTALEN)ZFN．2009(Xanthamonas)(transcriptionactivator-likeeffectorTALE)DNA[46]．2010TALEN[7]、、、、、[8]．TALENZFN．2012TALEN《》(Science)[9]．ZFNTALENDNAFok．2013clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)/CRISPR-associated(Cas)9、、[10]．CRISPR、CRISPR/CasCRISPR/Cas9．Prog.Biochem.Biophys.2013;40(8)2CRISPR1987K12[11]2002clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)[1214]．CRISPR．2005CRISPR(spacer)[1517]．2006CRISPRRNAiFig.1SchematicofCRISPR/Cas9mediatedDNAdouble-strandbreak1CRISPR/Cas9TracrRNA、RNaseCas9pre-RNATracrRNA、crRNACas9．RNasePre-crRNACas9tracrRNAtracrRNACas9Cas1Cas2Csn2CRISPRDNAPAM1CRISPR/CasCRISPR/Cas(1)．(StreptococcuspyogenesSF370)TypeCRISPR/Cas5'tracrRNACasCas9、Cas1、Cas2Csn23'CRISPR(spacers)(directrepeats)．5'3'692··,CRISPR/Cas92013;40(8)[18]．．2007Barrangou[19]CRSPR．2008Marraffini[20]CRISPRCRISPRCRISPR．CRISPR．3CRISPRCRISPRdbCRISPICRISPRCRISPRdbCRSPR(http//crispr.upsud.fr)[2122]．CRSPICasCRISPRsCPRISPRdb(http//crispi.geneouest.org/)．CRISPR90%40%CRISPR[23]．CRISPR21～72bpCRISPR．CRISPR(HaliangiumochraceumDSM14365)587[24]．CRISPR21～48bpCRISPR5'3'GTTT/gGAAAC．RNACas[14,2426]．CRISPRCas、、[27]．CasCRISPRRNACRISPR/Cas．CasCRISPRCRISPR．CRISPRCRISPRCasCas[26,2829]．CasCas[30]．CasCasCRISPR/CasCas1Cas2CRISPRRNA．CRISPR/CasType、Type、Type．TypeCRISPR/CasCas6Cas3[31]．CascrRNACRISPRCASCAD(CRISPRassociatedcomplexforantivirusdefense)CASCADDNACASCADcrRNADNARCas3RCas3[3234]．TypeCas9()crRNADNA[35]．Cas9NRucHNH．TypeCRISPR/CasCRISPR/CastracrRNA(trans-activatingcrRNA)RNaseCas9crRNA．tracrRNAcrRNARNACas9DNA[36]．TypeCas10RNATypeCASCAD[37]．Cas10crRNADNA．TypeTypeATypeB．CRISPR/CasTypeAmRNA[38]CRISPR/CasTypeB693··Prog.Biochem.Biophys.2013;40(8)CRISPRCRISPRRNA(pre-crRNA)CasRNARNAcrRNA[4348]．TypeCRISPR/CascrRNACas9RNasetracrRNA[36]．CRISPR[44,49]．CRISPR[4546,48,5051]．Fig.2NewspcacerofCRISPRacqusition2CRISPRTypeCRISPR/CasDNA[20]．CRISPR/Cas．CRISPR/Cas．TypeTypeTypeType[39]．2090CRISPR/CasCRISPR/Cas、[24,2829,40]．4CRISPR/CasCRISPR/CasCRISPRCRIPSR()CRISPR/Cas．CRISPR(spacer)DNADNACRRSPR5'(repeats)．CRISPR5'3'．protospacerprotospacer5'3'PAM(protospaceradjacentmotifs)2～5protospacer1～4[41]．DNAPAMPAMprotospacerCRISPR5'(2)．Cas1Cas2[19,42]．TypeCRISRP/CasCsn2[19,43]．CasCRISPRPAMCCCCCCCCCCCCCCCCCCCCCCCCCTTTTTTTTTTTTTGGGGCGGGGGGGGGGGGGGGGGGGGGGAAAAAAAAAAAAA5'3'5'3'694··,CRISPR/Cas92013;40(8)CRISPR/CascrRNACasDNADNAcrRNADNA．crRNA(spacer)DNA[19,52]spacerprotospacer[3435,53]．5CRISPRCRISPR/Cas．crRNADNAcrRNApre-crRNACRISPR/Cas．CRISPR．crRNAspacer5'3'repeat．DNAcrRNADNA(protopacer)“protospacer”crRNA“protospacer”repeat“protospacer”DNACRISPR/Cas[54]．CRRISP/CasCRISPRspacerXop411Xop411protospacerPAM(proto-spaceradjacentmotifs)DNAprotospacerPAMCRISPR/CasDNA[53]．CRISPR(spacer)PAMCRISPRDNADNA．6CRISRP/CasCRISPR/Cas．CRISPR/Cas．CRISPR[55]．CRISPR/CascrRNACasPAMprotospacer．CRISPR/Cas(engineeredendonucleaseEEN)．CRISPR/CasTypecrRNAtracrRNACas9．(StreptococcuspyogenesSF370)Type、、[5658]．ENNMartinTypeCRISPR/Cas9[5960]．tracrRNAtracrRNA5'crRNA3'(13bp)crRNA．tracrRNAcrRNAtracrRNAcrRNARNA(guideRNAgRNA)gRNAtracrRNAcrRNA(3)．Cas9NRuvCHNHRuvCHNHCas9PAM5'PAM3～8．2013MITZhangCRISPR/Cas9293TEMX1PVALBNero2ATh[58]．crRNARNase．《》695··Prog.Biochem.Biophys.2013;40(8)Fig.3SchematicofgRNA(chimericRNA)guideCas9site-specificcleavageinanimalgenome3gRNA(chemricRNA)Cas9(Science)Mali[57]CRISPR/Cas9．CRISPR/Cas9293TK652DNA()．ZhangZhanggRNAcrRNAtracrRNAMalicrRNAtracrRNAgRNA．NatureBiotechnologyCellResearchMaligRNA[56,61]．MaligRNA3'tracrRNAcrRNAtracrRNA．3'gRNA．ZFNTALEN[6264]CRISPR/Cas9．CRISPR/Cas9crRNA:tracrRNAgRNA．gRNAgRNA．gRNA．Cas9Cas9Cas9NC．ZhangCas9NLSCas9[58]．MaliCas9CCRSIPR/Cas9[57]．Shen[65]Cas9NNCCas9RNARNACas9:gRNACMVPolyAU6RNARNANLS-Cas9-NLS696··,CRISPR/Cas92013;40(8)Cas9NCas932．Cas9NLS“”．Cas9CCas9．6．1CRISPR/Cas9ZFNTALENCRISPR/Cas9．CRISRP/Cas9DNA(NHEJ)(HR)．[57]NHEJ．Cas9NHEJ．Ding[66]CellStemCelliPS、CRISPR/Cas9TALEN2．iPS[6770]．iPSiPSCRISPR/Cas9iPS．ZNF、TALENCRISPR/Cas9．CRISPR/Cas9iPS．6．2CRISPR/Cas91997“”．、．、、．(10－7)．ZFNTALENCRSPR/Cas9．ZFNTALEN[7174]CRISPR/Cas9．(singlenucleotidepolymorphismSNP)IFG22SNPIGF2[75]MSTN3'SNPMSTN[76]．SNP．SNP．SNP．SNP．ZFN、TALENCRISPR/Cas9CRISPR/Cas9．CRISPR/Cas98bpTALEN1/125bpZFN500bp[77]CRISPR/Cas9．CRISPR/Cas9ZFNTALENCas9DNACas9Cas9．ZFNTALENCRISPR/Cas9100%DNADNA．697··Prog.Biochem.Biophys.2013;40(8)ZFNTALENDNAZFNTALENDNAZFNTALEN．CRISPR/Cas9PAM(NGG)PAM11bp14bp(PAM+)CRISPR/Cas9．14bpDNACRISPR/Cas9．CRISPR/Cas9()[78]SNP．ZNFTALENZFNTALENDNA．ZFNTALENDNA(VP16KRAB)[7981]．Qi[82]CRIPSR/Cas9．Cas9DNAgRNA．RNA100%．