Development of a highly efficient base editing system for Lactobacilli to improve probiotics and dissect essential functions.

Lactobacilli play essential roles in the food industry and have a significant potential as probiotics and therapeutic agents. Genomic and genetic information has increasingly accumulated and been linked to their various functions, to which transgenic approaches are being performed to verify crucial genes. In order to reasonably develop more useful strains, beneficial traits need to be introduced into any given strains and enhanced or combined based on such genotype characterization. However, for practical use as probiotics or foods, organisms with transgene are hardly acceptable. Here, we have introduced the base editing Target-AID system specifically for Lactobacilli, enabling precise installation of point mutations without donor DNA and at multiple genomic loci simultaneously. Lactiplantibacillus plantarum has been successfully engineered to reduce production of imidazole propionate, which has been reported to be associated with type 2 diabetes by impairing glucose tolerance and insulin signaling. Additionally, this system enabled transient knock-out of an essential gene, such as one involved in cell division, resulting in severe filamentous cell phenotype. This demonstrates Target-AID is a promising genetic tool for Lactobacilli and can accelerate both applied and fundamental research. KEY POINTS: • Efficient and multiplexable cytosine base editing established in Lactobacilli. • Edited Lactobacillus reducing imidazole propionate associated with the risk of type 2 diabetes. • Transient knock-out and dissection of an essential gene function.