谷氨酸棒杆菌中基于CRISPR/Cas9的多位点碱基编辑系统的优化

作为新型的基因组编辑工具,碱基编辑技术结合了CRISPR/Cas系统的定位功能和碱基脱氨酶的编辑功能,可实现特定位点的碱基突变,具有不产生双链DNA断裂,无需外源模板且不依赖染色体DNA同源重组的优势。目前,研究者们已在重要的工业生产菌株谷氨酸棒杆菌(Corynebacterium glutamicum)中开发了多种碱基编辑工具,并实现了两基因和三基因的同时编辑。文中针对谷氨酸棒杆菌中基于CRISPR/Cas9的多位点碱基编辑系统中存在的不足(多重sgRNA结构烦琐、重复序列干扰、更换靶点困难等),采用多种策略进行优化,并比较碱基编辑效率:首先优化基于单独启动子/终止子的多重sgRNA表达框,通过构建框架质粒,并结合Golden Gate连接方法,加速靶点更换,避免重复序列干扰,虽然该方法 sgRNA结构烦琐,但编辑效率最高;同时,开发基于Type ⅡCRISPR crRNA阵列、tRNA加工的多重gRNA表达框,这两种形式均只需要一个启动子和一个终止子序列,极大地简化了表达框结构,虽然编辑效率均出现下降,但仍具有一定的实用性。该研究丰富了谷氨酸棒杆菌的基因组编辑工具,为该菌株的遗传改...

Optimization of CRISPR/Cas9-based multiplex base editing in Corynebacterium glutamicum

As a new CRISPR/Cas-derived genome engineering technology,base editing combines the target specificity of CRISPR/Cas and the catalytic activity of nucleobase deaminase to install point mutations at target loci without generating DSBs,requiring exogenous template,or depending on homologous recombination.Recently,researchers have developed a variety of base editing tools in the important industrial strain Corynebacterium glutamicum,and achieved simultaneous editing of two and three genes.However,the multiplex base editing based on CRISPR/Cas9 is still limited by the complexity of multiple sgRNAs,interference of repeated sequence and difficulty of target loci replacement.In this study,multiplex base editing in C.glutamicum was optimized by the following strategies.Firstly,the multiple sgRNA expression cassettes based on individual promoters/terminators was optimized.The target loci can be introduced and replaced rapidly by using a template plasmid and Golden Gate method,which also avoids the interference of repeated sequence.Although the multiple sgRNAs structure is still complicated,the editing efficiency of this strategy is the highest.Then,the multiple gRNA expression cassettes based on TypeⅡCRISPR crRNA arrays and tRNA processing were developed.The two strategies only require one single promoter and terminator,and greatly simplify the structure of the expression cassette.Although the editing efficiency has decreased,both methods are still applicable.Taken together,this study provides a powerful addition to the genome editing toolbox of C.glutamicum and facilitates genetic modification of this strain.