Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) microorganisms. They are widely used in the fermentation of food products for thousands of years. As human gut commensal bacteria, they can colonize the gastrointestinal (GI) tract to maintain gut health by eliciting beneficial host immune responses and combating pathogen infections. The role of LAB in fermenting foods, producing metabolites, and promoting human health makes them attractive bacterial chassis to develop next-generation probiotics. However, the development of genetic tools for genome engineering of LAB has lagged behind its significance. The proposed research will develop a genome engineering approach to construct genetic “landing pads” in LAB, which can lay a solid foundation for the development of probiotics with multiple therapeutic functions.