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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Base editing is a CRISPR/Cas-derived genome engineering technology that introduces point mutations without double-strand breaks or exogenous templates.
• Recent advancements have introduced base editing tools to Corynebacterium glutamicum, enabling simultaneous editing of multiple genes.
• Existing CRISPR/Cas9 multiplex base editing in C. glutamicum faces limitations due to complex sgRNA expression, repeated sequence interference, and challenges in target locus replacement.

Purpose of the Study:

• To optimize multiplex base editing strategies in Corynebacterium glutamicum.
• To overcome limitations of current multiplex base editing approaches in C. glutamicum, including sgRNA complexity and target locus replacement.
• To enhance the genome editing toolbox for C. glutamicum to facilitate genetic modification.

Main Methods:

• Optimization of multiple sgRNA expression cassettes using individual promoters/terminators.
• Rapid introduction and replacement of target loci via a template plasmid and Golden Gate method.
• Development of multiple gRNA expression cassettes utilizing Type II CRISPR crRNA arrays and tRNA processing.

Main Results:

• The strategy using individual promoters/terminators and Golden Gate method achieved the highest editing efficiency, despite complex sgRNA structures.
• Strategies employing Type II CRISPR crRNA arrays and tRNA processing simplified expression cassette structure by using a single promoter and terminator.
• While editing efficiency decreased with the simplified strategies, they remain applicable for multiplex base editing in C. glutamicum.

Conclusions:

• This study presents optimized multiplex base editing strategies for Corynebacterium glutamicum.
• The developed methods address limitations in sgRNA expression and target locus replacement, enhancing genome engineering capabilities.
• These advancements provide valuable tools for the genetic modification of C. glutamicum, an important industrial strain.