Advances in CRISPR-Based Genetic Engineering of Bifidobacterium.

Bifidobacterium plays a vital role in gut health, immunity, and metabolism, yet natural strains show limited native functional capabilities and genetic manipulability. Recent advances in genome editing, especially CRISPR-based systems, have provided novel approaches for engineering Bifidobacterium, though strain-specific variability and restriction-modification barriers remain substantial constraints. This review outlines the evolution from traditional homologous recombination to CRISPR/Cas technologies, highlighting applications of endogenous and exogenous systems, including CRISPRi/a and base editing. In preclinical and food biotechnology contexts, genetically engineered strains have shown potential as targeted delivery vehicles and functional probiotics. Despite these achievements, challenges such as strain variability, restriction-modification barriers, and biosafety concerns persist. Where genetic accessibility can be established, the integration of CRISPR technology with synthetic biology may enable more precise gene regulation and could support the development of next-generation engineered Bifidobacterium-based platforms.