Exploiting Endogenous Cas9-Based Genome Editing of Lacticaseibacillus rhamnosus GG (LGG) for Precision Food Fermentation and Live Biotherapeutics

Lacticaseibacillus rhamnosus GG (LGG) is one of the most studied probiotic strains and is widely used in both food and therapeutic industries. With its safe and health-beneficial properties, LGG is an ideal candidate for genome modification to enhance its functionalities for food, industrial and pharmaceutical applications. However, precise and efficient genome engineering tools for LGG are unavailable. In this study, we developed a novel genome engineering tool for LGG based on its endogenous type II-A CRISPR-Cas9 system. By employing a validated protospacer adjacent motif (PAM), a customized single guide RNA (sgRNA) expression cassette and a homologous repair template, we successfully reprogrammed the native CRISPR-Cas9 system for targeted genome modifications. Using this method, we successfully constructed a fucose-negative LGG incapable of growing on L-fucose and a lactose-positive LGG. The lactose-positive LGG exhibited significantly improved growth in milk compared to its parental strain LGG, which is unable to ferment lactose, attributed to its ability to metabolize lactose efficiently. Moreover, this strain showed robust growth during yogurt fermentation and maintained viability during cold storage for at least three weeks. These findings highlight the potential of the lactose-positive LGG as an improved culture for dairy industry and a functional probiotic for food and therapeutic applications.