CRISPR/Cas9-Based Genome Editing Platform for Companilactobacillus crustorum to Reveal the Molecular Mechanism of Its Probiotic Properties

Companilactobacillus crustorum usually serves as a starter culture for the food industry. Recent studies revealed that this species also possesses probiotic properties. Genome engineering, including point mutation or gene deletion, is desired to understand the mechanisms of its probiotic and fermentation properties. To tackle the hurdle in genetic manipulation in C. crustorum, here, we established a fast and easy CRISPR/Cas9-based platform for precise genome editing in this species. The platform includes two CRISPR/Cas9 systems and a CRISPR/Cas9-based editing system. Using the developed methods, we were able to knockout 12 genes in C. crustorum by deleting a fragment located in the open reading frames. The editing efficiency ranged from 14.3 to 100%. Moreover, we developed a CRISPR-assisted cytidine base-editing system, enabling programmed C to T conversion in the chromosome for gene inactivation or point mutation. To further exploit this platform, we investigated the role of nine putative bacteriocin-encoding genes and found that bacteriocins BM173 and BM1157 mostly contributed to the antimicrobial activity of C. crustorum MN047 against Staphylococcus aureus and Escherichia coli. In addition, the regulation of bacteriocin expression was also revealed to be linked with the quorum-sensing modulator luxS. This work will dramatically accelerate the genetic engineering of C. crustorum and close-related species.