# CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

## TL;DR

This study uses CRISPR to investigate efflux pumps in Mycobacterium abscessus, revealing their roles in drug resistance and virulence.

## Contribution

Developed optimized CRISPR-Cas9 tools for M. abscessus and identified key efflux pumps critical for drug resistance and virulence.

## Key findings

- Different efflux pumps have distinct roles in drug resistance and survival in Galleria mellonella larvae.
- The MAB_2806-2807 operon is vital for drug resistance and virulence in M. abscessus.
- CRISPR-based tools enable efficient genetic manipulation of M. abscessus for molecular research.

## Abstract

Mycobacterium abscessus, 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 M. abscessus. In this study, we modified the method used to prepare M. abscessus 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 M. abscessus and investigated their roles in drug resistance and virulence. We found that different efflux pumps play distinct roles in drug resistance and survival in Galleria mellonella larvae. Finally, we confirmed that MAB_2806‐2807, involved in transportation of triacylglycerides, is vital for the drug resistance and virulence of M. abscessus. The molecular biology tools developed in this study will facilitate molecular research on M. abscessus. In addition, the study on efflux pumps might provide new targets for the development of new drugs and treatment regimens for M. abscessus infection.

Hundreds of efflux pumps have been identified in Mycobacterium abscessus; however, their contributions to drug resistance and virulence remain undefined. Here, optimized CRISPR‐Cas9‐assisted tools were developed for efficient genetic manipulation of this pathogen and used to systematically generate knockout mutants of 66 efflux pump genes, each individually characterized. The MAB_2806–2807 operon was identified as the most critical determinant of antimicrobial resistance and virulence in M. abscessus. This study establishes a robust genome‐editing platform for M. abscessus research and enhances our understanding of efflux pump‐mediated drug resistance and pathogenicity.