食品与发酵工业 >

2019 , Vol. 45 >Issue 19: 1 - 7

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.021520

研究报告

谷氨酸棒状杆菌CRISPR-Cpf1/ssDNA基因组编辑系统优化

  • 王婷 ,
  • 马洪坤 ,
  • 赵桂红 ,
  • 蔡柠匀 ,
  • 张德志 ,
  • 陈宁
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  • 1(天津科技大学 生物工程学院,天津,300457)
    2(代谢控制发酵技术国家地方联合工程实验室(天津科技大学),天津,300457)
    3(教育部工业发酵微生物重点实验室(天津科技大学),天津,300457)
    4(天津市微生物代谢与发酵过程控制技术工程中心,天津,300457)

修回日期: 2019-07-18

  网络出版日期: 2019-11-15

基金资助

国家重点研发计划(2018YFA0900304); 工业微生物优良菌种选育与发酵技术公共服务平台项目(17PTGCCX00190)

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Optimization of a CRISPR-Cpf1/ssDNA genome editing system for Corynebacterium glutamicum

  • WANG Ting ,
  • MA Hongkun ,
  • ZHAO Guihong ,
  • CAI Ningyun ,
  • ZHANG Dezhi ,
  • CHEN Ning
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  • 1(College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
    2(National and Local United Engineering Lab of Metabolic Control Fermentation Technology(Tianjin University of Science and Technology), Tianjin 300457, China)
    3(Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education(Tianjin University of Science and Technology), Tianjin 300457, China)
    4(Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process Control, Tianjin 300457, China)

Revised date: 2019-07-18

  Online published: 2019-11-15

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摘要

谷氨酸棒状杆菌作为重要的微生物细胞工厂,基因组修饰已经成为调节目标代谢物的首要途径。为了提高基因定点突变的编辑效率,建立了高效省时的CRISPR-Cpf1/ssDNA基因组编辑系统。利用卡那霉素抗性作为筛选标记,构建了1株严谨的单链模式菌M-1,用于验证与统计编辑效率。首先,采用强启动子Ptuf,优化了切割效率;其次,利用重组酶RecT和合理长度与添加量的ssDNA,优化了重组效率。实验结果显示：在启动子Ptuf和RecT的双重作用下,采用滞后链(70 bp、12.5 μg)优化组合方式,使基因编辑效率提升至(80±5.7)%。由RecT介导的CRISPR-Cpf1/ssDNA基因组编辑系统可在很大程度上加快谷氨酸棒状杆菌代谢工程改造。

关键词： CRISPR-Cpf1; ssDNA; 谷氨酸棒状杆菌; 优化; 基因组编辑

本文引用格式

王婷 , 马洪坤 , 赵桂红 , 蔡柠匀 , 张德志 , 陈宁 . 谷氨酸棒状杆菌CRISPR-Cpf1/ssDNA基因组编辑系统优化[J]. 食品与发酵工业, 2019 , 45(19) : 1 -7 . DOI: 10.13995/j.cnki.11-1802/ts.021520

Abstract

Corynebacterium glutamicum is an important microbial cell factory, and genomic modification has become a primary way to regulate target metabolites. In order to improve the editing efficiency of site-directed mutation, an efficient and time-saving CRISPR-Cpf1/ssDNA genome editing system was established. The kanamycin resistance was used as a screening marker to construct a rigorous ssDNA model strain M-1 to verify and calculate editing efficiency. Firstly, strong promoter Ptuf was adopted to optimize cutting efficiency. Secondly, the recombination efficiency was optimized by recombinase RecT and reasonable length and addition amount of ssDNA. The results showed that under the dual action of promoter Ptuf and RecT, the gene editing efficiency increased to (80±5.7)% using the optimized combination of lagging strands (70 bp and 12.5 μg). Overall, the RecT-mediated CRISPR-Cpf1/ssDNA genome editing system can greatly accelerate the metabolic engineering modification of C. glutamicum.

Key words： CRISPR-Cpf1; ssDNA; Corynebacterium glutamicum; optimization; genome editing

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