谷氨酸棒状杆菌CRISPR-Cpf1/ssDNA基因组编辑系统优化

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

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(70bp、12.5μg)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。

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 .