Application and Research Prospects of CRISPR/Cas Gene Editing Technology in Lactic Acid Bacteria.

Lactic acid bacteria (LAB) are pivotal microorganisms in the food industry. Current approaches for functional gene validation and trait improvement in LAB primarily rely on traditional gene editing and homologous recombination techniques. These methods are often cumbersome, inefficient, and time-consuming, hindering the rapid and precise customization of strains. This limitation has, to some extent, constrained the rapid selection and industrial application of functional LAB strains. The engineering of LAB through gene editing technologies has significantly advanced both fundamental and applied research. Among these, CRISPR/Cas gene editing has successfully achieved precise modification of multiple genes in various LAB species. Compared to conventional methods, it offers superior editing efficiency and lower operational costs, opening new avenues for functional gene identification and genetic improvement in LAB. However, the application of exogenous CRISPR/Cas systems in LAB faces technical challenges such as high off-target rates, chromosomal abnormalities, and cytotoxicity. The development of endogenous CRISPR/Cas-based editing tools for LAB provides novel pathways for precise regulation, rational design, and flexible application. This paper first outlines the structural components and mechanistic principles of CRISPR/Cas gene editing tools. It then explores the research progress and applications of both endogenous and exogenous CRISPR/Cas systems in LAB. Finally, it provides an outlook on the future application of CRISPR/Cas gene editing technology in LAB, offering a reference for its implementation in this field. The advent of gene editing technologies has significantly propelled functional gene validation and trait improvement in lactic acid bacteria (LAB), thereby advancing both fundamental research and industrial applications. Notably, the CRISPR/Cas system has emerged as a transformative tool enabling precise genetic modification in diverse LAB species, offering marked improvements in editing efficiency and cost reduction relative to conventional approaches. CRISPR/Cas-based editing strategies in LAB are broadly classified into exogenous and endogenous systems. Exogenous systems operate independently of the host's native immune repertoire, conferring the advantages of broad strain applicability and high editing efficiency. These systems have been successfully deployed for functional gene characterization, metabolic pathway engineering, such as augmenting antimicrobial production, and probiotic safety enhancement via virulence gene deletion. Conversely, endogenous systems leverage the intrinsic CRISPR/Cas machinery of LAB, offering superior biocompatibility and minimized off-target risks. Notable applications include precise gene knockout and integration using the native Type I-E system in