CRISPRi-mediated validation of candidate Mycobacterium abscessus drug targets during host infection

Mycobacterium abscessus (Mab) is a multidrug-resistant nontuberculous mycobacterium that causes debilitating TB-like pulmonary infections for which effective treatment options are lacking. Poor in vivo 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 in vivo, we exploited CRISPRi (CRi) gene silencing in multiple mouse infection models. Inducible silencing of ftsZ_Mab , a previously validated target, and three predicted targets (leuS_Mab , folP_Mab , fusA_Mab ) confirmed their essentiality in vitro. We then assessed the in vivo 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 Mab burden. However, in C57BL/6N mice, the degree of Mab clearance varied among targets, suggesting that immune pressure may influence the outcome of CRi-mediated gene silencing. Notably, repression of fusA_Mab yielded a larger decline in mycobacterial burden in C57BL/6N mice despite a lower level of gene silencing in vitro, consistent with enhanced vulnerability of this target. Overall, this study demonstrated that ftsZ_Mab, leuS_Mab , folP_Mab , and fusA_Mab are essential for Mab growth in vitro 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 Mab infections.