Utilization of a CRISPRi-based challenge model to reveal temporally dependent gene essentiality in intracellular .

Abstract:

(Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters , the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. Using a library comprising ~20,000 sgRNAs covering >96% of Mtb open reading frames, we identified genes required for growth within the changing immune microenvironment. Mutant depletion patterns varied across immune environments sampled at 2, 4, and 6 weeks post-infection, which revealed a weighted dependency on cell wall biosynthesis genes early and the reliance on cholesterol catabolism and iron acquisition across all time points. Functional validation of three genes-, , and -confirmed their temporal significance . This screen provides increased resolution of the differential metabolic vulnerabilities in Mtb in the evolving immune environments during infection, stressing the temporal nature of conditional essentiality .