Comprehensive mapping of essential-gene interactions in M. tuberculosis

Abstract Mycobacterium tuberculosis , the causative agent of tuberculosis, remains the world’s deadliest pathogen 1 . Despite intensive study, many of its genes remain uncharacterized, which limits the insight that can be gained into tuberculosis biology and constrains drug development. Genetic interaction (GI) mapping is a powerful strategy to define functional relationships between genes by quantifying how the phenotypic effects of combined perturbations deviate from expectations based on single-gene perturbations 2,3 . Here we develop a dual-sgRNA CRISPR-interference platform that enables high-throughput GI mapping in mycobacteria. We address key technical challenges in library construction and data interpretation by incorporating unique barcodes for each sgRNA pair and implementing a robust statistical pipeline for GI detection. Focusing on essential genes, we generate comprehensive GI maps in the model mycobacterium Mycobacterium smegmatis and the pathogen M. tuberculosis . These maps recapitulate known biology, uncover thousands of previously unrecognized interactions and identify functional connections between essential genes and drug targets. By generating AlphaFold3-based protein–protein interaction predictions 4 for all gene pairs in our GI maps, we show that a subset of GIs is likely to reflect underlying physical associations. Validation of selected GIs confirms both novel biological relationships and opportunities for synergistic targeting. Overall, this work establishes a generalizable framework for dual-gene perturbation and defines the architecture of essential-gene networks in mycobacteria, advancing both our understanding of tuberculosis biology and the rational design of combination therapies.