生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (4): 106-.doi: 10.3969/j.issn.2095-1736.2020.04.106

• 技术方法 • 上一篇    下一篇

优化大肠杆菌CRISPR/Cas9基因编辑系统及应用

  

  1. 1. 天津科技大学 生物工程学院, 天津 310018； 2. 中国科学院 天津工业生物技术研究所,天津 300308； 3. 中国科学院 系统微生物工程重点实验室, 天津 300308
  • 出版日期:2020-08-18 发布日期:2020-08-10
  • 通讯作者: 张学礼，研究员，研究方向为微生物代谢工程，E-mail: zhang_xl@tib.cas.cn；朱欣娜，副研究员，研究方向为微生物代谢工程，E-mail: zhu_xn@tib.cas.cn
  • 作者简介:邵梦瑶，硕士研究生，研究方向为微生物代谢工程，E-mail: 815765734@qq.com
  • 基金资助:
    国家自然科学基金面上项目；CyaA*腺苷环化酶突变解除葡萄糖对木糖转运阻遏的机制 （No. 31870058）

Optimization of Escherichia coli CRISPR/Cas9 gene editing system and its application

  1. 1. College of Biotechnology, Tianjin Unisversity of Science and Technology, Tianjin 310018；
     2. Tianjin Institute of Industrial Biotechonology, Chinese Academy of Sciences, Tianjin 300308；
     3. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
  • Online:2020-08-18 Published:2020-08-10

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摘要： CRISPR/Cas9基因编辑系统目前已经广泛地应用于大肠杆菌工程菌株的构建，若能构建出一种能连续进行基因编辑的CRISPR/Cas9系统，将极大地提高大肠杆菌工程菌株构建的效率。设计并构建了具有自剪切功能的供体质粒pV4，优化了双质粒CRISPR/Cas9基因编辑系统，实现了基因的连续敲除或整合。pV4质粒，在原始供体质粒placZ的骨架区域添加3个元件：针对供体质粒上氯霉素基因cat的N20-gRNA序列；Ptrc启动子,用于表达cat-N20-gRNA；lacIq序列，用于表达LacI蛋白调控Ptrc启动子。pV4供体系列质粒（敲除或整合）实现基因编辑后，cat-N20-gRNA在IPTG的诱导表达引导Cas9蛋白对供体质粒进行自剪切，从而方便下一轮基因的编辑。将pV4系列质粒（敲除或整合）应用于产衣康酸工程菌株的构建，连续敲除ptsI、iclR及整合cad基因，基因编辑结果显示，优化后的双质粒CRISPR/Cas9基因编辑系统，能快速消除供体质粒，实现基因的连续敲除或整合，节约了基因操作的时间，有广泛的工程菌株构建应用前景。

关键词: CRISPR/Cas9, 基因编辑, 大肠杆菌, 自剪切功能

Abstract: CRISPR/Cas9 gene editing system has been widely used in the construction of Escherichia coli engineering strains. If a continuous gene editing-based CRISPR/Cas9 system can be constructed, the construction efficiency of E. coli engineering strains will be greatly improved. In this study, a donor plasmid pV4, with self-cutting function was constructed to optimize the existing double plasmid CRISPR/Cas9 gene editing system in the laboratory, and to realize the continuous knockout or integration of the gene. Three elements were added to the skeleton region of the original donor plasmid placZ: the N20-gRNA sequence of chloramphenicol gene(Cat) on the donor plasmid; Ptrc promoter; lacIq sequence. After gene editing with pV4 series plasmids(knockout or integration), these elements were induced by IPTG and with the help of Cas9 protein to achieve self-cutting of donor plasmids. The optimized pV4 series plasmids(knockout or integration) were used to engineer a strain to produce itaconic, and the result showed that this new CRISPR/Cas9 gene editing system can quickly perform continuous knockout or integration of genes, which is a broad prospect of construction and application of engineering strains.

Key words: CRISPR/Cas9, gene editing, Escherichia coli, self-cutting

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