The rapid emergence and spread of drug resistance in Mycobacterium tuberculosis has become a global health burden. Drug resistance in pathogens is often caused by mutations in core metabolic pathways. The disruptions in those pathways lead to fitness costs and collateral vulnerabilities in cells.

Using whole genome CRISPR interference, we identified genes that exhibit increased sensitivity to transcriptional inhibition, revealing both shared and strain-specific collateral vulnerabilities across genetically diverse drug-resistant M. tuberculosis. Notably, tRNA synthetases emerged as consistently more vulnerable across multiple drug-resistant genotypes, highlighting their potential as promising therapeutic targets. The rifampicin resistance mutation (RpoB^S450L) had unique vulnerabilities to the transcriptional repression of sulphur metabolism.

These results provide insight into the functional consequences of drug resistance and highlight new avenues for combating drug-resistant M. tuberculosis.