碱基编辑器的开发及其在细菌基因组编辑中的应用
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摘要
碱基编辑器是近两年发展起来的新型基因组编辑工具,它将碱基脱氨酶的催化活性和CRISPR/Cas系统的靶向特异性进行结合,催化DNA或RNA链上特定位点的碱基发生脱氨基反应,进而完成碱基的替换。碱基编辑器分为DNA和RNA碱基编辑器两大类,其中DNA碱基编辑器分为两种:胞嘧啶碱基编辑器和腺嘌呤碱基编辑器;前者可以实现胞嘧啶到胸腺嘧啶的转换,而后者则可以将腺嘌呤突变为鸟嘌呤。由于DNA碱基编辑器不会造成DNA的双链断裂(DSB),也不依赖于宿主的非同源末端修复和同源重组途径,因此,大大减少了DSB相关的编辑副产物,如小片段插入或缺失等。基于CRISPR/Cas系统的RNA碱基编辑器,可以实现RNA链上腺嘌呤核苷到次黄苷的转换。本文对不同类型碱基编辑器的开发过程、适用范围和编辑特点等进行梳理,并对其在细菌基因组编辑中的应用进行了介绍;最后简要探讨了细菌中碱基编辑器的缺点以及将来可能的研究方向。
Base editors are novel genome-editing tools developed in the past two years that comprisefusions between a catalytically disabled CRISPR/Cas endonuclease and a base deaminase to deaminate theexocyclic amine of the target bases, thereby leading to base substitutions in DNA or RNA. Two classes ofbase editors have been developed, namely DNA base editors and RNA base editors. Two types of DNAeditors have been described: cytosine base editors(CBEs) convert C to T and adenine base editors(ABEs)convert A to G. Base editors do not create double-strand DNA breaks(DSBs) and do not rely on cellularnon-homologous end joining(NHEJ) and homology-directed repair(HDR), so they minimize thegeneration of DSB-associated by products, such as small insertions or deletion(indels). RNA base editorsbased on CRISPR/Cas systems could achieve adenosine conversion to inosine. In this review, wesummarize the development process, scope of application and editing features of base editors and highlighttheir recent applications in bacterial genome editing. Finally, we will also briefly discuss limitations andfuture directions of base editors for applications in bacteria.
Base editors are novel genome-editing tools developed in the past two years that comprisefusions between a catalytically disabled CRISPR/Cas endonuclease and a base deaminase to deaminate theexocyclic amine of the target bases, thereby leading to base substitutions in DNA or RNA. Two classes ofbase editors have been developed, namely DNA base editors and RNA base editors. Two types of DNAeditors have been described: cytosine base editors(CBEs) convert C to T and adenine base editors(ABEs)convert A to G. Base editors do not create double-strand DNA breaks(DSBs) and do not rely on cellularnon-homologous end joining(NHEJ) and homology-directed repair(HDR), so they minimize thegeneration of DSB-associated by products, such as small insertions or deletion(indels). RNA base editorsbased on CRISPR/Cas systems could achieve adenosine conversion to inosine. In this review, wesummarize the development process, scope of application and editing features of base editors and highlighttheir recent applications in bacterial genome editing. Finally, we will also briefly discuss limitations andfuture directions of base editors for applications in bacteria.
引文
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[43]Wettengel J,Reautschnig P,Geisler S,et al.Harnessing human ADAR2 for RNA repair-Recoding a PINK1 mutation rescues mitophagy[J].Nucleic Acids Research,2017,45(5):2797-2808
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[47]Montiel-González MF,Vallecillo-Viejo I,Yudowski GA,et al.Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(45):18285-18290
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[49]Hess GT,Tycko J,Yao D,et al.Methods and applications of CRISPR-mediated base editing in eukaryotic genomes[J].Molecular Cell,2017,68(1):26-43
[50]Kim JS.Precision genome engineering through adenine and cytosine base editing[J].Nature Plants,2018,4(3):148-151
[51]Eid A,Alshareef S,Mahfouz MM.CRISPR base editors:genome editing without double-stranded breaks[J].Biochemical Journal,2018,475(11):1955-1964
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[2]Knott GJ,Doudna JA.CRISPR-Cas guides the future of genetic engineering[J].Science,2018,361(6405):866-869
[3]Shah SA,Erdmann S,Mojica FJM,et al.Protospacer recognition motifs:mixed identities and functional diversity[J].RNA Biology,2013,10(5):891-899
[4]Makarova KS,Wolf YI,Alkhnbashi OS,et al.An updated evolutionary classification of CRISPR-Cas systems[J].Nature Reviews Microbiology,2015,13(11):722-736
[5]Koonin EV,Makarova KS,Zhang F.Diversity,classification and evolution of CRISPR-Cas systems[J].Current Opinion in Microbiology,2017,37:67-78
[6]Hsu PD,Lander ES,Zhang F.Development and applications of CRISPR-Cas9 for genome engineering[J].Cell,2014,157(6):1262-1278
[7]Deltcheva E,Chylinski K,Sharma CM,et al.CRISPR RNAmaturation by trans-encoded small RNA and host factor RNase III[J].Nature,2011,471(7340):602-607
[8]Jinek M,Chylinski K,Fonfara I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science,2012,337(6096):816-921
[9]Paquet D,Kwart D,Chen A,et al.Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9[J].Nature,2016,533(7601):125-129
[10]Qi LS,Larson MH,Gilbert LA,et al.Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression[J].Cell,2013,152(5):1173-1183
[11]La Russa MF,Qi LS.The new state of the art:Cas9 for gene activation and repression[J].Molecular and Cellular Biology,2015,35(22):3800-3809
[12]Xu XS,Qi LS.A CRISPR-dCas toolbox for genetic engineering and synthetic biology[J].Journal of Molecular Biology,2019,431(1):34-47
[13]Komor AC,Kim YB,Packer MS,et al.Programmable editing of a target base in genomic DNA without double-stranded DNAcleavage[J].Nature,2016,533(7603):420-424
[14]Gaudelli NM,Komor AC,Rees HA,et al.Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage[J].Nature,2017,551(7681):464-471
[15]Rees HA,Liu DR.Base editing:precision chemistry on the genome and transcriptome of living cells[J].Nature Reviews Genetics,2018,19(12):770-788
[16]Cox DBT,Gootenberg JS,Abudayyeh OO,et al.RNA editing with CRISPR-Cas13[J].Science,2017,358(6366):1019-1027
[17]Harris RS,Petersen-Mahrt SK,Neuberger MS.RNA editing enzyme APOBEC1 and some of its homologs can act as DNAmutators[J].Molecular Cell,2002,10(5):1247-1253
[18]Mol CD,Arvai AS,Sanderson RJ,et al.Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor:protein mimicry of DNA[J].Cell,1995,82(5):701-708
[19]Nishida K,Arazoe T,Yachie N,et al.Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems[J].Science,2016,353(6305):aaf8729
[20]Komor AC,Zhao KT,Packer MS,et al.Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity[J].Science Advances,2017,3(8):eaao4774
[21]Kim YB,Komor AC,Levy JM,et al.Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions[J].Nature Biotechnology,2017,35(4):371-376
[22]Hu JH,Miller SM,Geurts MH,et al.Evolved Cas9 variants with broad PAM compatibility and high DNA specificity[J].Nature,2018,556(7699):57-63
[23]Nishimasu H,Shi X,Ishiguro S,et al.Engineered CRISPR-Cas9nuclease with expanded targeting space[J].Science,2018,361(6408):1259-1262
[24]Wang X,Li JN,Wang Y,et al.Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion[J].Nature Biotechnology,2018,36(10):946-949
[25]Ma YQ,Zhang JY,Yin WJ,et al.Targeted AID-mediated mutagenesis(TAM)enables efficient genomic diversification in mammalian cells[J].Nature Methods,2016,13(12):1029-1035
[26]Gehrke JM,Cervantes O,Clement MK,et al.An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities[J].Nature Biotechnology,2018,36(10):977-982
[27]Li XS,Wang Y,Liu YJ,et al.Base editing with a Cpf1-cytidine deaminase fusion[J].Nature Biotechnology,2018,36(4):324-327
[28]Zheng YX,Lorenzo C,Beal PA.DNA editing in DNA/RNAhybrids by adenosine deaminases that act on RNA[J].Nucleic Acids Research,2017,45(6):3369-3377
[29]Wolf J,Gerber AP,Keller W.TadA,an essential tRNA-specific adenosine deaminase from Escherichia coli[J].The EMBOJournal,2002,21(14):3841-3851
[30]Kim J,Malashkevich V,Roday S,et al.Structural and kinetic characterization of Escherichia coli TadA,the wobble-specific tRNA deaminase[J].Biochemistry,2006,45(20):6407-6416
[31]Matthews MM,Thomas JM,Zheng YX,et al.Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity[J].Nature Structural&Molecular Biology,2016,23(5):426-433
[32]Gerber AP,Keller W.An adenosine deaminase that generates inosine at the wobble position of tRNAs[J].Science,1999,286(5442):1146-1149
[33]Grunebaum E,Cohen A,Roifman CM.Recent advances in understanding and managing adenosine deaminase and purine nucleoside phosphorylase deficiencies[J].Current Opinion in Allergy and Clinical Immunology,2013,13(6):630-638
[34]Shi K,Carpenter MA,Banerjee S,et al.Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B[J].Nature Structural&Molecular Biology,2017,24(2):131-139
[35]Macbeth MR,Schubert HL,van Demark AP,et al.Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing[J].Science,2005,309(5740):1534-1539
[36]Losey HC,Ruthenburg AJ,Verdine GL.Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA[J].Nature Structural&Molecular Biology,2006,13(2):153-159
[37]Hua K,Tao XP,Zhu JK.Expanding the base editing scope in rice by using Cas9 variants[J].Plant Biotechnology Journal,2018.DOI:10.1111/pbi.12993
[38]Yang L,Zhang XH,Wang LR,et al.Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants[J].Protein&Cell,2018,9(9):814-819
[39]Nishikura K.Functions and regulation of RNA editing by ADARdeaminases[J].Annual Review of Biochemistry,2010,79:321-349
[40]Tan MH,Li Q,Shanmugam R,et al.Dynamic landscape and regulation of RNA editing in mammals[J].Nature,2017,550(7675):249-254
[41]Bass BL,Weintraub H.An unwinding activity that covalently modifies its double-stranded RNA substrate[J].Cell,1988,55(6):1089-1098
[42]Wong SK,Sato S,Lazinski DW.Substrate recognition by ADAR1and ADAR2[J].RNA,2001,7(6):846-858
[43]Wettengel J,Reautschnig P,Geisler S,et al.Harnessing human ADAR2 for RNA repair-Recoding a PINK1 mutation rescues mitophagy[J].Nucleic Acids Research,2017,45(5):2797-2808
[44]Stafforst T,Schneider MF.An RNA-deaminase conjugate selectively repairs point mutations[J].Angewandte Chemie International Edition,2012,51(44):11166-11169
[45]Vogel P,Schneider MF,Wettengel J,et al.Improving site-directed RNA editing in vitro and in cell culture by chemical modification of the guideRNA[J].Angewandte Chemie International Edition,2014,53(24):6267-6271
[46]Vogel P,Moschref M,Li Q,et al.Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs[J].Nature Methods,2018,15(7):535-538
[47]Montiel-González MF,Vallecillo-Viejo I,Yudowski GA,et al.Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(45):18285-18290
[48]Montiel-González MF,Vallecillo-Viejo IC,Rosenthal JJC.An efficient system for selectively altering genetic information within m RNAs[J].Nucleic Acids Research,2016,44(21):e157
[49]Hess GT,Tycko J,Yao D,et al.Methods and applications of CRISPR-mediated base editing in eukaryotic genomes[J].Molecular Cell,2017,68(1):26-43
[50]Kim JS.Precision genome engineering through adenine and cytosine base editing[J].Nature Plants,2018,4(3):148-151
[51]Eid A,Alshareef S,Mahfouz MM.CRISPR base editors:genome editing without double-stranded breaks[J].Biochemical Journal,2018,475(11):1955-1964
[52]Komor AC,Badran AH,Liu DR.Editing the genome without double-stranded DNA breaks[J].ACS Chemical Biology,2018,13(2):383-388
[53]Wu WY,Yang YH,Lei HT.Progress in the application of CRISPR:From gene to base editing[J].Medicinal Research Reviews,2018.DOI:10.1002/med.21537
[54]May A.Base editing on the rise[J].Nature Biotechnology,2017,35(5):428-429
[55]Plosky BS.CRISPR-mediated base editing without DNAdouble-strand breaks[J].Molecular Cell,2016,62(4):477-478
[56]Bjerke JN,Beardslee PC,McNaughton BR.Recent advances in CRISPR base editing:from A to RNA[J].Biochemistry,2018,57(6):886-887
[57]Seo H,Kim JS.Towards therapeutic base editing[J].Nature Medicine,2018,24(10):1493-1495
[58]Arazoe T,Kondo A,Nishida K.Targeted nucleotide editing technologies for microbial metabolic engineering[J].Biotechnology Journal,2018,13(9):1700596
[59]Banno S,Nishida K,Arazoe T,et al.Deaminase-mediated multiplex genome editing in Escherichia coli[J].Nature Microbiology,2018,3(4):423-429
[60]Zheng K,Wang Y,Li N,et al.Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion[J].Communications Biology,2018,1(1):32
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