Development and application of CRISPR/Cas9-assisted genome-editing tools for comprehensive investigation the role of efflux pumps in Mycobacterium abscessus

Using CRISPR/Cas9-assisted genome-editing tools, we constructed 68 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. To investigate the role of efflux pumps in the virulence of M. abscessus, M. abscessus mutants were transformed with barcode-containing integrative plasmids and then mixed prior to infecting G. mellonella larvae for 3 and 5 days. The M. abscessus cells were then collected from infected G. mellonella and analyzed using next generation sequencing.