CRISPR‐based genome editing reveals the roles of efflux pumps in <i>Mycobacterium abscessus</i>

<i>Mycobacterium abscessus</i>, one of the most antimicrobial-resistant bacteria, is increasingly recognized as the cause of infections that are difficult to treat. Novel genetic manipulation tools are required to elucidate the biology, pathogenesis, and antibiotic resistance mechanisms of <i>M. abscessus</i>. In this study, we modified the method used to prepare <i>M. abscessus</i> electrocompetent cells to achieve efficient transformation, and then optimized the CRISPR-Cas9-assisted genome-editing tools to allow efficient genetic manipulation. Using these tools, we constructed 66 efflux pump mutants of <i>M. abscessus</i> and investigated their roles in drug resistance and virulence. We found that different efflux pumps play distinct roles in drug resistance and survival in <i>Galleria mellonella</i> larvae. Finally, we confirmed that MAB_2806-2807, involved in transportation of triacylglycerides, is vital for the drug resistance and virulence of <i>M. abscessus</i>. The molecular biology tools developed in this study will facilitate molecular research on <i>M. abscessus</i>. In addition, the study on efflux pumps might provide new targets for the development of new drugs and treatment regimens for <i>M. abscessus</i> infection.