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The functional characterization of the extensive repertoire of genes encoding virulence factors and drug targets has been largely restricted by the lack of powerful genetic tools. In this study, we evaluated the performances of a Tet-OFF system, previously optimized for\n            <jats:italic>M. tuberculosis<\/jats:italic>\n            and\n            <jats:italic>M. smegmatis<\/jats:italic>\n            . Fluorescence reporter\n            <jats:italic>M. abscessus<\/jats:italic>\n            strains were developed where mCherry was under the control of the Tet-OFF regulatory system on an integrative plasmid. The addition of anhydrotetracycline (ATc) correlated with a decrease in fluorescence intensity not only in solid and broth medium but also in colony biofilms, for both smooth and rough variants of\n            <jats:italic>M. abscessus<\/jats:italic>\n            . Next, unmarked\n            <jats:italic>mmpL3<\/jats:italic>\n            conditional knockdown mutants were engineered in both smooth and rough\n            <jats:italic>M. abscessus<\/jats:italic>\n            . Biochemical studies indicated that the addition of ATc was associated with a dose-dependent decrease in (i) the production of the MmpL3 protein, (ii) the biosynthesis of trehalose dimycolate and mycolylated arabinogalactan, (iii) bacterial viability on agar and liquid medium as well as in biofilms. Importantly, intravenous injection of ATc in zebrafish embryos infected with the rough\n            <jats:italic>mmpL3<\/jats:italic>\n            conditional mutant impeded bacterial growth and correlated with a significant gain in embryo survival. These results suggest that\n            <jats:italic>mmpL3<\/jats:italic>\n            is essential for\n            <jats:italic>M. abscessus<\/jats:italic>\n            growth\n            <jats:italic>in vitro<\/jats:italic>\n            and in the infected host, thus validating MmpL3 as an attractive drug target. Together, this study demonstrates the potential of ATc-dependent repression to modulate the expression of\n            <jats:italic>M. abscessus<\/jats:italic>\n            genes in the infected host.\n          <\/jats:p>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              <jats:italic>Mycobacterium abscessus<\/jats:italic>\n              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\n              <jats:italic>M. abscessus<\/jats:italic>\n              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\n              <jats:italic>mmpL3<\/jats:italic>\n              , encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of\n              <jats:italic>mmpL3<\/jats:italic>\n              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\n              <jats:italic>M. abscessus<\/jats:italic>\n              gene during host infection.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.02836-23","type":"journal-article","created":{"date-parts":[[2023,10,13]],"date-time":"2023-10-13T15:33:05Z","timestamp":1697211185000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":20,"title":["Silencing essential gene expression in\n            <i>Mycobacterium abscessus<\/i>\n            during infection"],"prefix":"10.1128","volume":"11","author":[{"given":"Yves-Marie","family":"Boudehen","sequence":"first","affiliation":[{"name":"Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Universit\u00e9 de Montpellier , Montpellier, 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