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;lacI^q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。
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;lacI^q 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.
作者
邵梦瑶
路福平
朱欣娜
张学礼
SHAO Meng-yao;LU Fu-ping;ZHU Xin-na;ZHANG Xue-Li(College of Biotechnology,Tianjin Unisversity of Science and Technology,Tianjin 310018;Tianjin Institute of Industrial Biotechonology,Chinese Academy of Sciences,Tianjin 300308;Key Laboratory of Systems Microbial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)
出处
《生物学杂志》
CAS
CSCD
北大核心
2020年第4期106-110,114,共6页
Journal of Biology
基金
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)。
