Fig. 1: Essential role of IleRS in mycobacterial growth and survival revealed by CRISPRi silencing.

A Schematic diagram of CRISPRi-mediated silencing of IleRS. B Growth of M. abscessus on 7H10 solid medium containing 500 ng/mL ATC at 37 °C and M. marinum on 7H10 containing 200 ng/mL ATC at 30 °C, starting from an initial dilution gradient of OD₆₀₀ = 0.1. C Growth curves of M. abscessus and M. marinum in 7H9-OADC medium supplemented with 500 ng/mL ATC at 37 °C or 200 ng/mL ATC at 30 °C, respectively (n = 3). For the purpose of this study, “KD” refers to gene silencing via CRISPRi. D Silencing of IleRS in M. abscessus and M. marinum results in impaired carbon and nitrogen source utilization and reduced growth under acidic and nutrient-poor conditions (n = 3). The figure shows relative growth rates of empty vector strains (PLJR962_KD) and IleRS-silenced strains (IleRS_KD) during the logarithmic growth phase under ATC treatment compared to untreated conditions, expressed as OD₆₀₀^+ATC / OD₆₀₀^−ATC. For M. abscessus, relative growth rates were calculated at selected time points (36 h or 48 h), and for M. marinum, at 3d or 5d. The full set of observed time points and growth curves are shown in Supplementary Figs. S1 and S2. An text asterisk (*) indicates severe growth defects with failure to reach the normal plateau phase; absence of an asterisk indicates only a delay in reaching the plateau phase. Error bars represent mean ± SD. Statistical significance was determined by two-way ANOVA. Differences were considered statistically significant at P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****). “ns” denotes no significant difference.