High-Fidelity Cytosine Base Editing in a GC-Rich Corynebacterium glutamicum with Reduced DNA Off-Target Editing Effects
Yu Been Heo, Gue‐Ho Hwang, Seok Won Kang, Sangsu Bae, Han Min Woo
Abstract
Rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we identified the DNA off-targets for single and multiple genome engineering of the industrial bacterium Corynebacterium glutamicum using whole-genome sequencing. Further, we developed the high-fidelity (HF)-CBE with significantly reduced off-targets with comparable efficiency and precision. We believe that our DNA off-target analysis and the HF-CBE can promote CRISPR-assisted genome engineering over conventional gene manipulation tools by providing a markerless genetic tool without need for a foreign DNA donor.