CRISPR/Cas9-Mediated Genome Editing for , a Novel Species with Clinical, Animal, and Plant-Associated Isolates.

As one of the most widespread groups of Gram-negative bacteria, bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. is a novel species of with clinical, animal, and plant-associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time-consuming and laborious endeavor. Here, by using the CRISPR-Cas system, a versatile gene-editing tool with a two-plasmid strategy was developed for a native strain isolated from the model organism silkworm () gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic-related gene, , was knocked out, resulting in the slow growth of the deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked-in exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel species.