Optimization of CRISPR/Cas9-based multiplex base editing in <i>Corynebacterium glutamicum</i>.

Hui LU; Qi Zhang; Sili YU; Yu Wang; Ming Kang; Shuangyan Han; Ye Liu; Meng Wang

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

Author: Hui LU ¹ ; Qi ZHANG ² ; Sili YU ² ; Yu WANG ² ; Ming KANG ³ ; Shuangyan HAN ¹ ; Ye LIU ² ; Meng WANG ²
Author Information

1. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China.
2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
3. School of Life Sciences, Hebei University, Baoding 071002, Hebei, China.
Publication Type:Journal Article
Keywords: CRISPR/Cas system; Corynebacterium glutamicum; base editing; multiple gRNAs
MeSH: CRISPR-Cas Systems/genetics*; Corynebacterium glutamicum/metabolism*; Gene Editing; Plasmids; RNA, Guide/metabolism*
From: Chinese Journal of Biotechnology 2022;38(2):780-795
CountryChina
Language:Chinese
Abstract: 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 Type Ⅱ CRISPR 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.