Silencing essential gene expression in during infection.

represents the most common rapidly growing mycobacterial pathogen in cystic fibrosis and is extremely difficult to eradicate. Essential genes are required for growth, often participate in pathogenesis, and encode valid drug targets for further chemotherapeutic developments. However, assessing the function of essential genes in remains challenging due to the limited spectrum of efficient genetic tools. Herein, we generated a Tet-OFF-based system allowing to knock down the expression of , encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of in planktonic cultures, in biofilms, and during infection in zebrafish embryos. Thus, in this study, we developed a robust and reliable method to silence the expression of any gene during host infection.