Harnessing CRISPR-Cas9 for Lactobacillus improvement in silage production: current knowledge and future perspectives.

High-quality silage is the cornerstone to sustainable livestock development and animal food production. As the core fermentation bacteria of silage, Lactobacillus directly regulates silage fermentation by producing lactic acid, enzymes, and other bioactive molecules. However, traditional screening methods for functional strains are labor-intensive and time-consuming. Recent advances in synthetic biology, particularly the development of CRISPR-Cas genome editing technology, offer a revolutionary approach to designing Lactobacillus strains with customized traits. This review systematically reviewed the importance of silage in sustainable agricultural development and the limitations of current silage preparation and promotion. It also discussed the application of strain engineering approaches in optimizing the phenotypic performance of Lactobacillus for better silage. Building on this, we reviewed the research progress of CRISPR-Cas9 gene editing in Lactobacillus and discussed how to leverage its high efficiency and precision to optimize the strain's traits for improved silage quality and functionality. CRISPR-Cas9 toolkits are expected to achieve directed evolution of strain performance, ultimately yielding next-generation silage microbial inoculants with multiple functions, adaptability to multiple substrates, and eco-friendly characteristics. The use of such innovative biotechnologies would facilitate resource-efficient utilization, promote animal performance and health for sustainable development in livestock production.