构建CRISPR-Cas9介导的耻垢分枝杆菌基因组高效删除系统
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摘要
【背景】耻垢分枝杆菌具有生长迅速和非致病性的特点,可作为结核分枝杆菌致病机理研究替代菌株和类固醇激素生产的工程菌,但目前耻垢分枝杆菌中缺乏高效率的基因组敲除方法。【目的】基于CRISPR-Cas9介导的定点、高效的DNA切割能力,构建耻垢分枝杆菌染色体DNA片段无痕敲除系统。【方法】构建了包含四环素诱导型启动子驱动的密码子优化的cas9基础载体pCas9101,在双侧同源臂长度约为1 kb条件下选用合适的gRNA表达模块,分别测试了对耻垢分枝杆菌mc2155染色体上的3β-羟基类固醇脱氢酶基因(MSMEG_5228,1 071 bp)和胆固醇降解基因簇(MSMEG_5990-MSMEG_6043,约48kb)敲除效率,使用相同大小的同源臂以经典p2NIL-pGOAL方法进行对照,并计算效率。【结果】使用CRISPR-Cas9方法对耻垢分枝杆菌mc2155的3β-羟基类固醇脱氢酶基因敲除效率为22%,胆固醇降解基因簇敲除效率也达到18%,两者连续敲除效率为4%。但对照p2NIL-pGOAL方法未能获得目标DNA片段敲除的菌株。【结论】本文建立的基于CRISPR-Cas9的耻垢分枝杆菌基因组无痕敲除系统显示出较高的敲除效率,该方法可为耻垢分枝杆菌后续研究提供快速高效的基因组操作方法。
[Background] For its fast growing and non-pathogenic property, Mycobacterium smegmatis is used as a model strain for pathogenic Mycobacterium tuberculosis and a workhorse to produce steroid hormones. But it lacks efficient genome deletion system. [Objective] In this study, we established a high efficiency genome deletion system in M. smegmatis assisted by CRISPR-Cas9. [Methods] The Cas9 backbone plasmid pCas9101 was constructed with a tetracycline-inducible codon-optimized cas9 expression operon. Approximate 1 kb flanked homologous arms and proper gRNA operon were used to construct vectors to study the deletion efficiency on 3β-hydroxysteroid dehydrogenase(MSMEG_5228, 1 071 bp) encoding gene and cholesterol degradation gene cluster(MSMEG_5990-MSMEG_6043, about 48 kb) in M. smegmatis mc2155. The classical p2 NIL-pGOAL method with same homologous arms was used as the control to delete the two same DNA segments in M. smegmatis mc2155. Their deletion efficiency were calculated and compared. [Results] When using CRISPR-Cas9 assisted method, 22% and 18% clones showed the expected deletions in MSMEG_5228 gene and cholesterol-degrading gene cluster, respectively. Even the sequential deletion efficiency on both segments can reach 4%. However, no expected deletion mutants were obtained in our experiments with p2 NIL-pGOAL method. [Conclusion] This CRISPR-Cas9 assisted system can facilitate genome deletion in M. smegmatis mc2155, and provide a fast and efficient genome manipulation approach for Mycobacterium in the future.
[Background] For its fast growing and non-pathogenic property, Mycobacterium smegmatis is used as a model strain for pathogenic Mycobacterium tuberculosis and a workhorse to produce steroid hormones. But it lacks efficient genome deletion system. [Objective] In this study, we established a high efficiency genome deletion system in M. smegmatis assisted by CRISPR-Cas9. [Methods] The Cas9 backbone plasmid pCas9101 was constructed with a tetracycline-inducible codon-optimized cas9 expression operon. Approximate 1 kb flanked homologous arms and proper gRNA operon were used to construct vectors to study the deletion efficiency on 3β-hydroxysteroid dehydrogenase(MSMEG_5228, 1 071 bp) encoding gene and cholesterol degradation gene cluster(MSMEG_5990-MSMEG_6043, about 48 kb) in M. smegmatis mc2155. The classical p2 NIL-pGOAL method with same homologous arms was used as the control to delete the two same DNA segments in M. smegmatis mc2155. Their deletion efficiency were calculated and compared. [Results] When using CRISPR-Cas9 assisted method, 22% and 18% clones showed the expected deletions in MSMEG_5228 gene and cholesterol-degrading gene cluster, respectively. Even the sequential deletion efficiency on both segments can reach 4%. However, no expected deletion mutants were obtained in our experiments with p2 NIL-pGOAL method. [Conclusion] This CRISPR-Cas9 assisted system can facilitate genome deletion in M. smegmatis mc2155, and provide a fast and efficient genome manipulation approach for Mycobacterium in the future.
引文
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[16]Song CW,Lee J,Lee SY.Genome engineering and gene expression control for bacterial strain development[J].Biotechnology Journal,2015,10(1):56-68
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[20]Liu DY,Qiu T,Ding XH,et al.Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR-Cas9 genomic editing technology[J].Hereditas,2016,38(8):756-764(in Chinese)刘丁源,邱婷,丁晓辉,等.快速构建多重sgRNA载体利用CRISPR-Cas9技术敲除拟南芥IAA2基因[J].遗传,2016,38(8):756-764
[21]Cobb RE,Wang YJ,Zhao HM.High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system[J].ACS Synthetic Biology,2015,4(6):723-728
[22]Huang H,Zheng GS,Jiang WH,et al.One-step high-efficiency CRISPR-Cas9-mediated genome editing in Streptomyces[J].Acta Biochimica et Biophysica Sinica,2015,47(4):231-243
[23]Jiang Y,Chen B,Duan CL,et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system[J].Applied&Environmental Microbiology,2015,81(7):2506-2514
[24]DiCarlo JE,Norville JE,Mali P,et al.Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems[J].Nucleic Acids Research,2013,41(7):4336-4343
[25]Wu YZ,Xu HJ,Bai YL,et al.Comparison of CRISRP-Cas9system and mazF-mediated method for large deletions in Saccharomyces cerevisiae[J].Acta Microbiologica Sinica,2017,57(11):1604-1611(in Chinese)吴玉珍,徐海津,白艳玲,等.CRISPR-Cas9系统与mazF介导的大片段删减法在酿酒酵母染色体大片段删减中的比较[J].微生物学报,2017,57(11):1604-1611
[26]Guo XV,Monteleone M,Klotzsche M,et al.Silencing essential protein secretion in Mycobacterium smegmatis by using tetracycline repressors[J].Journal of Bacteriology,2007,189(13):4614-4623
[27]Snapper SB,Melton RE,Mustafa S,et al.Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis[J].Molecular Microbiology,1990,4(11):1911-1919
[28]Lu ZQ,Xie PF,Qin ZJ.Promotion of markerless deletion of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor[J].Acta Biochimica et Biophysica Sinica,2010,42(10):717-721
[29]Pattanayak V,Lin S,Guilinger JP,et al.High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9nuclease specificity[J].Nature Biotechnology,2013,31(9):839-843
[2]Ma JY,Huang HB,Xie YC,et al.Biosynthesis of ilamycins featuring unusual building blocks and engineered production of enhanced anti-tuberculosis agents[J].Nature Communications,2017,8(1):391
[3]van Kessel JC,Hatfull GF.Recombineering in Mycobacterium tuberculosis[J].Nature Methods,2007,4(2):147-152
[4]Choudhary E,Lunge A,Agarwal N.Strategies of genome editing in mycobacteria:Achievements and challenges[J].Tuberculosis,2016,98:132-138
[5]Galán B,Uhía I,García-Fernández E,et al.Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons[J].Microbial Biotechnology,2017,10(1):138-150
[6]Alvarez E,Tavel E.Reche’rches sur le bacille’de Lustgarden[J].Archives de Physiologie Normalet Pathologique,1885,(6):303-321
[7]Reyrat JM,Kahn D.Mycobacterium smegmatis:an absurd model for tuberculosis?[J].Trends in Microbiology,2001,9(10):472-474
[8]Parish T,Stoker NG.Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABCmutant by gene replacement[J].Microbiology,2000,146(8):1969-1975
[9]Du QL,Fan XY,Mao JX,et al.Progression on genetic knockout tools in Mycobacterium[J].Hereditas,2012,34(7):857-862(in Chinese)杜庆林,樊祥宇,毛金校,等.分枝杆菌中基因敲除操作工具研究进展[J].遗传,2012,34(7):857-862
[10]Malaga W,Perez E,Guilhot C.Production of unmarked mutations in mycobacteria using site-specific recombination[J].FEMSMicrobiology Letters,2003,219(2):261-268
[11]Yao K.Investigation into the molecular mechanism of microbial sterol degradation and its metabolic engineering for the production of steroid pharmaceutical precursors[D].Shanghai:Doctoral Dissertation of East China University of Science and Technology,2014(in Chinese)姚抗.分枝杆菌甾醇代谢机制的解析以及其代谢工程改造应用于制备重要甾药中间体的研究[D].上海:华东理工大学博士学位论文,2014
[12]Doudna JA,Charpentier E.The new frontier of genome engineering with CRISPR-Cas9[J].Science,2014,346(6213):1258096
[13]Xiong BB,Zeng H,Liu YK,et al.New era of gene editing driven by CRISPR-Cas9[J].Microbiology China,2017,44(1):178-185(in Chinese)熊彬彬,曾虎,刘云坤,等.CRISPR-Cas9驱动的基因编辑新纪元[J].微生物学通报,2017,44(1):178-185
[14]Li SY,Zhao GP,Wang J.Enabling technologies in synthetic biology-DNA synthesis,assembly and editing[J].Chinese Journal of Biotechnology,2017,33(3):343-360(in Chinese)李诗渊,赵国屏,王金.合成生物学技术的研究进展--DNA合成、组装与基因组编辑[J].生物工程学报,2017,33(3):343-360
[15]Ran FA,Hsu PD,Wright J,et al.Genome engineering using the CRISPR-Cas9 system[J].Nature Protocols,2013,8(11):2281-2308
[16]Song CW,Lee J,Lee SY.Genome engineering and gene expression control for bacterial strain development[J].Biotechnology Journal,2015,10(1):56-68
[17]Mali P,Esvelt KM,Church GM.Cas9 as a versatile tool for engineering biology[J].Nature Methods,2013,10(10):957-963
[18]Kennedy EM,Cullen BR.Bacterial CRISPR-Cas DNAendonucleases:A revolutionary technology that could dramatically impact viral research and treatment[J].Virology,2015,479-480:213-220
[19]Jiang Y,Qian FH,Yang JJ,et al.CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum[J].Nature Communications,2017,8:15179
[20]Liu DY,Qiu T,Ding XH,et al.Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR-Cas9 genomic editing technology[J].Hereditas,2016,38(8):756-764(in Chinese)刘丁源,邱婷,丁晓辉,等.快速构建多重sgRNA载体利用CRISPR-Cas9技术敲除拟南芥IAA2基因[J].遗传,2016,38(8):756-764
[21]Cobb RE,Wang YJ,Zhao HM.High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system[J].ACS Synthetic Biology,2015,4(6):723-728
[22]Huang H,Zheng GS,Jiang WH,et al.One-step high-efficiency CRISPR-Cas9-mediated genome editing in Streptomyces[J].Acta Biochimica et Biophysica Sinica,2015,47(4):231-243
[23]Jiang Y,Chen B,Duan CL,et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system[J].Applied&Environmental Microbiology,2015,81(7):2506-2514
[24]DiCarlo JE,Norville JE,Mali P,et al.Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems[J].Nucleic Acids Research,2013,41(7):4336-4343
[25]Wu YZ,Xu HJ,Bai YL,et al.Comparison of CRISRP-Cas9system and mazF-mediated method for large deletions in Saccharomyces cerevisiae[J].Acta Microbiologica Sinica,2017,57(11):1604-1611(in Chinese)吴玉珍,徐海津,白艳玲,等.CRISPR-Cas9系统与mazF介导的大片段删减法在酿酒酵母染色体大片段删减中的比较[J].微生物学报,2017,57(11):1604-1611
[26]Guo XV,Monteleone M,Klotzsche M,et al.Silencing essential protein secretion in Mycobacterium smegmatis by using tetracycline repressors[J].Journal of Bacteriology,2007,189(13):4614-4623
[27]Snapper SB,Melton RE,Mustafa S,et al.Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis[J].Molecular Microbiology,1990,4(11):1911-1919
[28]Lu ZQ,Xie PF,Qin ZJ.Promotion of markerless deletion of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor[J].Acta Biochimica et Biophysica Sinica,2010,42(10):717-721
[29]Pattanayak V,Lin S,Guilinger JP,et al.High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9nuclease specificity[J].Nature Biotechnology,2013,31(9):839-843