CRISPRi-mediated validation of candidate <i>Mycobacterium abscessus</i> drug targets during host infection

<i>Mycobacterium abscessus</i> (<i>Mab</i>) is a multidrug-resistant nontuberculous mycobacterium that causes debilitating TB-like pulmonary infections for which effective treatment options are lacking. Poor <i>in vivo</i> drug efficacy may stem from altered vulnerability of drug targets driven by host-specific environmental conditions. To enable validation and prioritization of candidate drug targets <i>in vivo</i>, we exploited CRISPRi (CRi) gene silencing in multiple mouse infection models. Inducible silencing of <i>ftsZ_Mab</i> , a previously validated target, and three predicted targets (<i>leuS_Mab</i> , <i>folP_Mab</i> , <i>fusA_Mab</i> ) confirmed their essentiality <i>in vitro</i>. We then assessed the <i>in vivo</i> vulnerability of these targets in both immunocompetent C57BL/6N and immunodeficient NSG mice by assessing the impact of CRi silencing on pulmonary mycobacterial burden. In NSG mice, silencing of all four genes led to comparable decreases in <i>Mab</i> burden. However, in C57BL/6N mice, the degree of <i>Mab</i> clearance varied among targets, suggesting that immune pressure may influence the outcome of CRi-mediated gene silencing. Notably, repression of <i>fusA_Mab</i> yielded a larger decline in mycobacterial burden in C57BL/6N mice despite a lower level of gene silencing <i>in vitro</i>, consistent with enhanced vulnerability of this target. Overall, this study demonstrated that <i>ftsZ_Mab, leuS_Mab</i> , <i>folP_Mab</i> , and <i>fusA_Mab</i> are essential for <i>Mab</i> growth <i>in vitro</i> and, for the first time, validated their vulnerability to inhibition by CRi during infection. These data also identified potential context-dependent target vulnerabilities, which could inform the prioritization of bacterial drug targets and accelerate the development of effective therapeutics for <i>Mab</i> infections.