High-Fidelity Cytosine Base Editing in a GC-Rich Corynebacterium glutamicum with Reduced DNA Off-Target Editing...

Area of Science:

• Synthetic Biology
• Microbial Engineering
• Genome Editing

Background:

• Cytosine base editors (CBEs) are powerful tools for microbial cell factory programming.
• Standard CBEs can induce unintended DNA off-target mutations, limiting their application in industrial bacteria.
• Corynebacterium glutamicum, a GC-rich bacterium, serves as a model for industrial applications.

Purpose of the Study:

• To develop and validate high-fidelity CBEs for precise genome engineering in Corynebacterium glutamicum.
• To address the challenge of off-target mutations in bacterial genome editing.
• To enhance succinate production through sequential gene inactivation.

Main Methods:

• Utilized a CBE-STOP approach to introduce premature termination codons (PTCs) in target genes.
• Employed whole-genome sequencing (WGS) to identify and analyze DNA off-target mutations.
• Developed and tested high-fidelity CBE variants (pCoryne-YE1-BE3 and pCoryne-BE3-R132E) to reduce off-target effects.

Main Results:

• Achieved high base editing efficiencies (average 95.6%) for single-gene inactivation and up to 100% success for PTCs.
• Successfully constructed a strain with five sequentially inactivated genes for enhanced succinate production.
• Demonstrated significantly reduced sgRNA-independent off-target mutations using high-fidelity CBEs, with average on-target efficiency of 90.5%.

Conclusions:

• High-fidelity CBEs offer a precise and efficient CRISPR-assisted genome engineering tool for Corynebacterium glutamicum.
• This approach minimizes undesirable mutations, overcoming limitations of previous CBE technologies.
• The developed HF-CBEs provide a markerless genetic tool, advancing microbial engineering without foreign DNA donors.