Genome Editing Using the Endogenous Type I‐E CRISPR‐Cas System in Lactobacillus paracasei ATCC334

SUMMARY Lactobacillus paracasei ATCC334 is a well‐known beneficial strain that plays a crucial role in food industry and promotion of human health. However, despite its significance, our understanding of its gene functions remains limited due to obstacles in gene editing techniques. This gap hinders the full utilization and development of this beneficial bacterium. In this study, we targeted L. paracasei ATCC334 as editing chassis. Initially, bioinformatics tools were used to explore a type I‐E endogenous clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas system within L. paracasei ATCC334. We further analyzed its repeat sequences, spacer sequences, and leader sequence predicted the protospacer adjacent motif (PAM) recognized by this system. To validate our findings, we assessed the accuracy of potential PAM, evaluated the cutting activity of the endogenous CRISPR‐Cas system, and studied the impact of the artificial mini‐CRISPR array through plasmid interference and genome interference experiments. These results helped us to achieve successful gene knockout and gene integration. Finally, we engineered a strain capable of nicotine degradation. Our study provides valuable insights for the broader development and application of lactobacilli.