An Efficient CRISPR/Cas12e System for Genome Editing in .

CRISPR/Cas systems have been widely used in the precise and traceless genetic engineering of bacteria. 320 (SM320) is a Gram-negative bacterium with a low efficiency of homologous recombination but a strong ability to produce vitamin B. Here, a CRISPR/Cas12e-based genome engineering toolkit, CRISPR/Cas12eGET, was constructed in SM320. The expression level of CRISPR/Cas12e was tuned through promoter optimization and the use of a low copy plasmid to adjust Cas12e cutting activity to the low homologous recombination efficiency of SM320, resulting in improved transformation and precision editing efficiencies. Furthermore, the accuracy of CRISPR/Cas12eGET was improved by deleting the gene involved in NHEJ repair in SM320. This advance will be useful for metabolic engineering and basic research on SM320, and it further provides a platform to develop the CRISPR/Cas system in strains where the efficiency of homologous recombination is low.