Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

• Published in: Biotechnology and bioengineering Vol. 116; no. 11; pp. 3016 - 3029
• Main Authors: Wang, Yu, Liu, Ye, Li, Junwei, Yang, Yi, Ni, Xiaomeng, Cheng, Haijiao, Huang, Teng, Guo, Yanmei, Ma, Hongwu, Zheng, Ping, Wang, Meng, Sun, Jibin, Ma, Yanhe
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.11.2019
• Subjects: Adenine; Adenosine; Adenosine deaminase; Amino acid substitution; Amino acids; Bacteria; base editing; Corynebacterium glutamicum; CRISPR; Cytidine deaminase; Deactivation; Deoxyribonucleic acid; Dependence; Deregulation; DNA; DNA damage; Editing; Feedback inhibition; Gene sequencing; Genomes; gRNA design; Homologous recombination; Homology; Inactivation; Lysine; Metabolic engineering; Metabolism; nucleobase deaminase; PAM requirement; PAM requirement; CRISPR; gRNA design; nucleobase deaminase; base editing; Corynebacterium glutamicum
• ISSN: 0006-3592; 1097-0290; 1097-0290
• DOI: 10.1002/bit.27121

CRISPR/Cas9‐guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double‐stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome‐targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome‐targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9‐fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5‐bp editing window to 7‐bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user‐friendly online tool named gBIG was provided for designing guide RNAs for base editing‐mediated inactivation of given genes in any given sequenced genome (www.ibiodesign.net/gBIG). Wang and coworkers updated the base editing toolbox for Corynebacterium glutamicum by extending the targeting scope, editing window and base transition capability of base editors. A user‐friendly online tool named gBIG (www.ibiodesign.net/gBIG) was also provided for designing guide RNAs for base editing‐mediated inactivation of given genes in any given sequenced genome. With these updates, base editing can serve as an effective method for fast strain development.