谷氨酸棒杆菌碱基编辑的条件优化

Optimization of base editing in Corynebacterium glutamicum

摘要:

近年来，基于CRISPR/Cas9的碱基编辑技术因其具有不产生DNA双链断裂、无需外源DNA模板、不依赖宿主同源重组修复的优势，已经逐渐发展成为一种强大的基因组编辑工具，在动物、植物、酵母和细菌中得到了开发和应用。研究团队前期已在重要的工业模式菌株谷氨酸棒杆菌中开发了一种多元自动化的碱基编辑技术MACBETH，为进一步优化该方法，提高碱基编辑技术在谷氨酸棒杆菌中的应用效率，本研究首先在谷氨酸棒杆菌中构建了基于绿色荧光蛋白 (GFP) 的检测系统：将GFP基因的起始密码子ATG人工突变为ACG，GFP无法正常表达，当该密码子的C经编辑后恢复为T，即实现GFP蛋白的复活，结合流式细胞仪分析技术，可快速衡量编辑效率。然后，构建针对靶标位点的碱基编辑工具，经测试，该位点可成功被编辑，在初始编辑条件下碱基编辑效率为 (13.11±0.21)%。在此基础上，通过对不同培养基类型、诱导初始OD600、诱导时间、诱导物浓度进行优化，确定最优编辑条件是：培养基为CGXII，初始OD600为0.05，诱导时间为20 h，IPTG浓度为0.01 mmol/L。经过优化，编辑效率达到 (30.35±0.75)%，较初始条件提高了1.3倍。最后，选取原编辑条件下编辑效率较低的位点，进行了优化后编辑条件下的编辑效率评估，结果显示，不同的位点在最优编辑条件下的编辑效率提高了1.7–2.5倍，进一步证实该优化条件的有效性及通用性。研究结果为碱基编辑技术在谷氨酸棒杆菌中更好的应用提供了重要的参考价值。

Abstract:

In recent years, CRISPR/Cas9-mediated base editing has been developed to a powerful genome editing tool, providing advantages such as without introducing double-stranded DNA break, a donor template and relying on host homologous recombination repair pathway, and has been widely applied in animals, plants, yeast and bacteria. In previous study, our group developed a multiplex automated base editing method (MACBETH) in the important industrial model strain Corynebacterium glutamicum. In this study, to further optimize the method and improve the base editing efficiency in C.?glutamicum, we first constructed a green fluorescent protein (GFP) reporter-based detection system. The point mutation in the inactivated GFP protein can be edited to restore the GFP fluorescence. By combining with flow cytometry analysis, the base-editing efficiency can be quickly calculated. Then, the base editor with the target gRNA was constructed, and the editing efficiency with the initial editing condition was (13.11±0.21)%. Based on this result, the editing conditions were optimized and the result indicated that the best medium is CGXII, the best initial OD600 of induction is 0.05, the best induction time is 20 h, and the best IPTG concentration is 0.01 mmol/L. After optimization, the editing efficiency was improved to (30.35±0.75)%, which was 1.3-fold of that in initial condition. Finally, endogenous genomic loci of C. glutamicum were selected to assess if the optimized condition can improve genome editing in other loci. Editing efficiency of different loci in optimized condition were improved to 1.7–2.5 fold of that in original condition, indicating the effectiveness and versatility of the optimized condition. Our research will promote the better application of base editing technology in C. glutamicum.