CRISPR-Cas9系统的基因编辑工具的应用和改进
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摘要
基因编辑技术可以实现对特定目的基因的编辑,加速了基因功能的研究、疾病的研究与治疗、药物的开发等,是生命科学的重要研究手段。基于CRISPR-Cas9系统的基因编辑工具可改变基因特定位点的序列、调控基因表达水平、敲除基因、改变特定位点表观遗传修饰以及活细胞成像等,成功应用于多种动植细胞系和模式生物。本文就基于Cas9的基因编辑工具的现状和相关改进展开综述。
Genome editing technologies allow precise gene manipulation, which facilitate the understanding of gene function and molecular basis of diseases as well as the development of therapeutics and are becoming indispensable tools in biological research. The applications based on CRISPR-Cas9 systems are widely used in various plant and animal cell lines and model organisms and enable endogenous gene manipulation, including sequence alteration, regulation of gene expression, gene knockout, epigenetic marks alteration, and lived cell imaging, etc. Here we will briefly describe the mechanisms and development of current available Cas9-based gene editing tools.
Genome editing technologies allow precise gene manipulation, which facilitate the understanding of gene function and molecular basis of diseases as well as the development of therapeutics and are becoming indispensable tools in biological research. The applications based on CRISPR-Cas9 systems are widely used in various plant and animal cell lines and model organisms and enable endogenous gene manipulation, including sequence alteration, regulation of gene expression, gene knockout, epigenetic marks alteration, and lived cell imaging, etc. Here we will briefly describe the mechanisms and development of current available Cas9-based gene editing tools.
引文
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[11]Garneau JE, Dupuis ME, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010, 468(7320):67-71
[12]Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage foradaptiveimmunityinbacteria.ProcNatl AcadSci USA, 2012, 109(39):E2579-2586
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[31]ChoSW,KimS,Kim Y,etal. Analysisofoff-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res, 2014, 24(1):132-141
[32]Mali P, Aach J, Stranges PB, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol,2013, 31(9):833-838
[33]RanFA,HsuPD, WrightJ,etal.Genomeengineering using the CRISPR-Cas9 system. Nat Protoc, 2013, 8(11):2281-2308
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[36]LinS,StaahlBT, AllaRK,etal.Enhancedhomologydirected human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife, 2014, 3:e04766
[37]Dow LE, Fisher J, O'Rourke KP, et al. Inducible in vivo genomeeditingwithCRISPR-Cas9.NatBiotechnol,2015, 33(4):390-394
[38]Nihongaki Y, Kawano F, Nakajima T, et al. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat Biotechnol, 2015, 33(7):755-760
[39]HemphillJ,BorchardtEK,BrownK,etal.Optical control of CRISPR/Cas9 gene editing. J Am Chem Soc,2015, 137(17):5642-5645
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[43]Pawluk A, Amrani N, Zhang Y, et al. Naturally occurring off-switches for CRISPR-Cas9. Cell, 2016, 167(7):1829-1838 e1829
[44]RauchBJ,SilvisMR,HultquistJF,etal.Inhibitionof CRISPR-Cas9withbacteriophageproteins.Cell,2017,168(1-2):150-158 e110
[45]Komor AC,Kim YB,PackerMS,etal.Programmable editing of a target base in genomic DNA without doublestranded DNA cleavage. Nature, 2016, 533(7603):420-424
[46]Nishida K, Arazoe T, Yachie N, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, 2016, 353(6305):pii aaf8729
[47]Hess GT, Fresard L, Han K, et al. Directed evolution using dCas9-targetedsomatichypermutationinmammalian cells. Nat Methods, 2016, 13(12):1036-1042
[48]Ma Y,ZhangJ, Yin W,etal. Targeted AID-mediated mutagenesis(TAM)enables efficient genomic diversification in mammalian cells. Nat Methods, 2016, 13(12):1029-1035
[49]Gaudelli NM, Komor AC, Rees HA, et al. Programmable baseeditingof A*TtoG*CingenomicDNAwithout DNA cleavage. Nature, 2017, 551(7681):464-471
[50]Komor AC, Zhao KT, Packer MS, et al. Improved base excisionrepairinhibitionandbacteriophageMuGam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv, 2017, 3(8):eaao4774
[51]ReesHA,Komor AC, YehWH,etal.Improvingthe DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun,2017, 8:15790
[52]Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013, 152(5):1173-1183
[53]Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013, 154(2):442-451
[54]Thakore PI, D'Ippolito AM, Song L, et al. Highly specific epigenomeeditingbyCRISPR-Cas9repressorsfor silencingofdistalregulatoryelements.NatMethods,2015, 12(12):1143-1149
[55]MaederML,LinderSJ,CascioVM,etal.CRISPR RNA-guided activation of endogenous human genes. Nat Methods, 2013, 10(10):977-979
[56]Perez-PineraP,KocakDD,VockleyCM,etal.RNA-guidedgeneactivationbyCRISPR-Cas9-based transcriptionfactors.NatMethods,2013,10(10):973-976
[57]Cheng AW, Wang H, Yang H, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res, 2013, 23(10):1163-1171
[58]Chavez A,ScheimanJ, VoraS,etal.Highlyefficient Cas9-mediated transcriptional programming. Nat Methods,2015, 12(4):326-328
[59]Zalatan JG, Lee ME, Almeida R, et al. Engineering complexsynthetictranscriptionalprogramswithCRISPR RNA scaffolds. Cell, 2015, 160(1-2):339-350
[60]Konermann S, Brigham MD, Trevino AE, et al. Genomescale transcriptional activation by an engineered CRISPRCas9 complex. Nature, 2015, 517(7536):583-588
[61]Nihongaki Y, YamamotoS,KawanoF,etal.CRISPRCas9-based photoactivatable transcription system. Chem Biol, 2015, 22(2):169-174
[62]PolsteinLR,GersbachCA. Alight-inducibleCRISPR-Cas9 system for control of endogenous gene activation. Nat Chem Biol, 2015, 11(3):198-200
[63]Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol, 2015, 33(2):139-142
[64]Gao Y, Xiong X, Wong S, et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat Methods, 2016, 13(12):1043-1049
[65]Liu XS, Wu H, Ji X, et al. Editing DNA methylation in the mammalian genome. Cell, 2016, 167(1):233-247.e17
[66]Amabile A,Migliara A,CapassoP,etal.Inheritable silencingofendogenousgenesbyhit-and-runtargeted epigenetic editing. Cell, 2016, 167(1):219-232.e14
[67]Galonska C, Charlton J, Mattei AL, et al. Genome-wide trackingofdCas9-methyltransferasefootprints.Nat Commun, 2018, 9(1):597
[68]XuX, Tao Y,GaoX,etal. ACRISPR-basedapproach for targeted DNA demethylation. Cell Discov, 2016, 2:16009
[69]Morita S, Noguchi H, Horii T, et al. Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFvTET1catalyticdomainfusions.NatBiotechnol,2016,34(10):1060-1065
[70]LindhoutBI,FranszP,TessadoriF,etal.Livecell imagingofrepetitiveDNAsequencesviaGFP-tagged polydactyl zinc finger proteins. Nucleic Acids Res, 2007,35(16):e107
[71]Miyanari Y, Ziegler-Birling C, Torres-Padilla ME. Live visualizationofchromatindynamicswithfluorescent TALEs. Nat Struct Mol Biol, 2013, 20(11):1321-1324
[72]QinP,ParlakM,KuscuC,etal.Livecellimagingof low-and non-repetitive chromosome loci using CRISPRCas9. Nat Commun, 2017, 8:14725
[73]MaH,Naseri A,Reyes-GutierrezP,etal.Multicolor CRISPRlabelingofchromosomallociinhumancells.Proc Natl Acad Sci USA, 2015, 112(10):3002-3007
[74]MaH, TuLC,Naseri A,etal.Multiplexedlabelingof genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol, 2016, 34(5):528-530
[75]Morgan SL, Mariano NC, Bermudez A, et al. ManipulationofnucleararchitecturethroughCRISPR-mediated chromosomal looping. Nat Commun, 2017, 8:15993
[76]HaoN,ShearwinKE,DoddIB.ProgrammableDNA looping using engineered bivalent dCas9 complexes. Nat Commun, 2017, 8(1):1628
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[2]HilleF,CharpentierE.CRISPR-Cas:biology,mechanismsandrelevance.Philos TransRSocLondBBiol Sci, 2016, 371(1707):pii:20150196
[3]Labrie SJ, Samson JE, Moineau S. Bacteriophage resistance mechanisms. Nat Rev Microbiol, 2010, 8(5):317-327
[4]MarraffiniLA.CRISPR-Casimmunityinprokaryotes.Nature, 2015, 526(7571):55-61
[5]MohanrajuP,MakarovaKS,ZetscheB,etal.Diverse evolutionaryrootsandmechanisticvariationsofthe CRISPR-Cas systems. Science, 2016, 353(6299):aad5147
[6]vanderOostJ,JoreMM, WestraER,etal.CRISPRbasedadaptiveandheritableimmunityinprokaryotes.Trends Biochem Sci, 2009, 34(8):401-407
[7]MakarovaKS,HaftDH,BarrangouR,etal.Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol, 2011, 9(6):467-477
[8]Makarova KS, Wolf YI, Alkhnbashi OS, et al. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol, 2015, 13(11):722-736
[9]ShmakovS, AbudayyehOO,MakarovaKS,etal.Discovery and functional characterization of diverse class 2CRISPR-Cas systems. Mol Cell, 2015, 60(3):385-397
[10]Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificityofgenomemodification.NatBiotechnol,2014,32(6):577-582
[11]Garneau JE, Dupuis ME, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010, 468(7320):67-71
[12]Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage foradaptiveimmunityinbacteria.ProcNatl AcadSci USA, 2012, 109(39):E2579-2586
[13]Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337(6096):816-821
[14]DeveauH,BarrangouR,GarneauJE,etal.Phageresponse to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol, 2008, 190(4):1390-1400
[15]Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, et al.Short motif sequences determine the targets of the prokaryoticCRISPRdefencesystem.Microbiology,2009,155(Pt 3):733-740
[16]Amrani N, Gao XD, Liu P, et al. NmeCas9 is an intrinsicallyhigh-fidelitygenome-editingplatform.Genome Biol, 2018, 19(1):214
[17]Ibraheim R, Song CQ, Mir A, et al. All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidisCas9invivo.GenomeBiol,2018,19(1):137
[18]KimE,Koo T,ParkSW,etal.Invivogenomeediting withasmallCas9orthologuederivedfromCampylobacter jejuni. Nat Commun, 2017, 8:14500
[19]Lee CM, Cradick TJ, Bao G. The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells. Mol Ther, 2016, 24(3):645-654
[20]AndersC,NiewoehnerO,Duerst A,etal.Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 2014, 513(7519):569-573
[21]Huai C, Li G, Yao R, et al. Structural insights into DNA cleavage activation of CRISPR-Cas9 system. Nat Commun, 2017, 8(1):1375
[22]Jiang F, Taylor DW, Chen JS, et al. Structures of a CRISPRCas9 R-loop complex primed for DNA cleavage. Science,2016, 351(6275):867-871
[23]Jiang F, Zhou K, Ma L, et al. STRUCTURAL BIOLOGY.ACas9-guideRNAcomplexpreorganizedfortarget DNA recognition. Science, 2015, 348(6242):1477-1481
[24]JinekM,JiangF, TaylorDW,etal.StructuresofCas9endonucleasesrevealRNA-mediatedconformational activation. Science, 2014, 343(6176):1247997
[25]Nishimasu H, Ran FA, Hsu PD, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell,2014, 156(5):935-949
[26]Anders C, Bargsten K, Jinek M. Structural plasticity of PAMrecognitionbyengineeredvariantsoftheRNAguided endonuclease Cas9. Mol Cell, 2016, 61(6):895-902
[27]Hu JH, Miller SM, Geurts MH, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 2018, 556(7699):57-63
[28]Nishimasu H, Shi X, Ishiguro S, et al. Engineered CRISPRCas9nucleasewithexpandedtargetingspace.Science,2018, 361(6408):1259-1262
[29]Casini A,OlivieriM,PetrisG,etal. Ahighlyspecific SpCas9 variant is identified by in vivo screening in yeast.Nat Biotechnol, 2018, 36(3):265-271
[30]Fu Y, Sander JD, Reyon D, et al. Improving CRISPR-Cas nucleasespecificityusingtruncatedguideRNAs.Nat Biotechnol, 2014, 32(3):279-284
[31]ChoSW,KimS,Kim Y,etal. Analysisofoff-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res, 2014, 24(1):132-141
[32]Mali P, Aach J, Stranges PB, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol,2013, 31(9):833-838
[33]RanFA,HsuPD, WrightJ,etal.Genomeengineering using the CRISPR-Cas9 system. Nat Protoc, 2013, 8(11):2281-2308
[34]Tsai SQ, Wyvekens N, Khayter C, et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol, 2014, 32(6):569-576
[35]KimS,KimD,ChoSW,etal.Highlyefficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res, 2014,24(6):1012-1019
[36]LinS,StaahlBT, AllaRK,etal.Enhancedhomologydirected human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife, 2014, 3:e04766
[37]Dow LE, Fisher J, O'Rourke KP, et al. Inducible in vivo genomeeditingwithCRISPR-Cas9.NatBiotechnol,2015, 33(4):390-394
[38]Nihongaki Y, Kawano F, Nakajima T, et al. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat Biotechnol, 2015, 33(7):755-760
[39]HemphillJ,BorchardtEK,BrownK,etal.Optical control of CRISPR/Cas9 gene editing. J Am Chem Soc,2015, 137(17):5642-5645
[40]OakesBL,NadlerDC,Flamholz A,etal.Profilingof engineeringhotspotsidentifiesanallostericCRISPRCas9 switch. Nat Biotechnol, 2016, 34(6):646-651
[41]TangW,HuJH,LiuDR. Aptazyme-embeddedguide RNAs enable ligand-responsive genome editing and transcriptional activation. Nat Commun, 2017, 8:15939
[42]Harrington LB, Doxzen KW, Ma E, et al. A broad-spectruminhibitorofCRISPR-Cas9.Cell,2017,170(6):1224-1233 e1215
[43]Pawluk A, Amrani N, Zhang Y, et al. Naturally occurring off-switches for CRISPR-Cas9. Cell, 2016, 167(7):1829-1838 e1829
[44]RauchBJ,SilvisMR,HultquistJF,etal.Inhibitionof CRISPR-Cas9withbacteriophageproteins.Cell,2017,168(1-2):150-158 e110
[45]Komor AC,Kim YB,PackerMS,etal.Programmable editing of a target base in genomic DNA without doublestranded DNA cleavage. Nature, 2016, 533(7603):420-424
[46]Nishida K, Arazoe T, Yachie N, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, 2016, 353(6305):pii aaf8729
[47]Hess GT, Fresard L, Han K, et al. Directed evolution using dCas9-targetedsomatichypermutationinmammalian cells. Nat Methods, 2016, 13(12):1036-1042
[48]Ma Y,ZhangJ, Yin W,etal. Targeted AID-mediated mutagenesis(TAM)enables efficient genomic diversification in mammalian cells. Nat Methods, 2016, 13(12):1029-1035
[49]Gaudelli NM, Komor AC, Rees HA, et al. Programmable baseeditingof A*TtoG*CingenomicDNAwithout DNA cleavage. Nature, 2017, 551(7681):464-471
[50]Komor AC, Zhao KT, Packer MS, et al. Improved base excisionrepairinhibitionandbacteriophageMuGam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv, 2017, 3(8):eaao4774
[51]ReesHA,Komor AC, YehWH,etal.Improvingthe DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun,2017, 8:15790
[52]Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013, 152(5):1173-1183
[53]Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013, 154(2):442-451
[54]Thakore PI, D'Ippolito AM, Song L, et al. Highly specific epigenomeeditingbyCRISPR-Cas9repressorsfor silencingofdistalregulatoryelements.NatMethods,2015, 12(12):1143-1149
[55]MaederML,LinderSJ,CascioVM,etal.CRISPR RNA-guided activation of endogenous human genes. Nat Methods, 2013, 10(10):977-979
[56]Perez-PineraP,KocakDD,VockleyCM,etal.RNA-guidedgeneactivationbyCRISPR-Cas9-based transcriptionfactors.NatMethods,2013,10(10):973-976
[57]Cheng AW, Wang H, Yang H, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res, 2013, 23(10):1163-1171
[58]Chavez A,ScheimanJ, VoraS,etal.Highlyefficient Cas9-mediated transcriptional programming. Nat Methods,2015, 12(4):326-328
[59]Zalatan JG, Lee ME, Almeida R, et al. Engineering complexsynthetictranscriptionalprogramswithCRISPR RNA scaffolds. Cell, 2015, 160(1-2):339-350
[60]Konermann S, Brigham MD, Trevino AE, et al. Genomescale transcriptional activation by an engineered CRISPRCas9 complex. Nature, 2015, 517(7536):583-588
[61]Nihongaki Y, YamamotoS,KawanoF,etal.CRISPRCas9-based photoactivatable transcription system. Chem Biol, 2015, 22(2):169-174
[62]PolsteinLR,GersbachCA. Alight-inducibleCRISPR-Cas9 system for control of endogenous gene activation. Nat Chem Biol, 2015, 11(3):198-200
[63]Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol, 2015, 33(2):139-142
[64]Gao Y, Xiong X, Wong S, et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat Methods, 2016, 13(12):1043-1049
[65]Liu XS, Wu H, Ji X, et al. Editing DNA methylation in the mammalian genome. Cell, 2016, 167(1):233-247.e17
[66]Amabile A,Migliara A,CapassoP,etal.Inheritable silencingofendogenousgenesbyhit-and-runtargeted epigenetic editing. Cell, 2016, 167(1):219-232.e14
[67]Galonska C, Charlton J, Mattei AL, et al. Genome-wide trackingofdCas9-methyltransferasefootprints.Nat Commun, 2018, 9(1):597
[68]XuX, Tao Y,GaoX,etal. ACRISPR-basedapproach for targeted DNA demethylation. Cell Discov, 2016, 2:16009
[69]Morita S, Noguchi H, Horii T, et al. Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFvTET1catalyticdomainfusions.NatBiotechnol,2016,34(10):1060-1065
[70]LindhoutBI,FranszP,TessadoriF,etal.Livecell imagingofrepetitiveDNAsequencesviaGFP-tagged polydactyl zinc finger proteins. Nucleic Acids Res, 2007,35(16):e107
[71]Miyanari Y, Ziegler-Birling C, Torres-Padilla ME. Live visualizationofchromatindynamicswithfluorescent TALEs. Nat Struct Mol Biol, 2013, 20(11):1321-1324
[72]QinP,ParlakM,KuscuC,etal.Livecellimagingof low-and non-repetitive chromosome loci using CRISPRCas9. Nat Commun, 2017, 8:14725
[73]MaH,Naseri A,Reyes-GutierrezP,etal.Multicolor CRISPRlabelingofchromosomallociinhumancells.Proc Natl Acad Sci USA, 2015, 112(10):3002-3007
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