Development of a highly efficient base editing system forLactobacillito improve probiotics and dissect essential functions

Lactobacilli play essential roles in the food industry and are increasingly explored for their potential as probiotics and therapeutic agents. Beneficial strains are primarily isolated from various natural sources including healthy human bodies, and undergo rigorous characterization and safety evaluations. 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. 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. Additionally, this system enabled transient knock-out of an essential gene, such as one involved in cell division showing severe filamentous cell phenotype, providing a unique approach for dissecting essential gene function. Importance This work provides highly efficient and multiplexable base editing system that installs precise point mutations in the genomes of the two major Lactobacilli strains. As the advanced CRISPR technology so-called non-cleaving genome editing, base editing is less toxic and does not integrate any foreign DNA into the genomes. Our approaches pave the way for dissecting and improving probiotics and food-grade microbes, ultimately creating better human health.