Genome scale CRISPRi reveals both shared and strain-specific vulnerabilities in genetically diverse drug-resistant strains of Mycobacterium tuberculosis

The global health burden caused by Mycobacterium tuberculosis is aggravated by the emergence and spread of drug resistance. Mutations that cause drug resistance can have collateral effects that increase the vulnerability of downstream pathways to inhibition. Here, using genome scale CRISPR interference we identified collateral effects associated with different drug-resistant genotypes in M. tuberculosis. We demonstrate that drug resistance generated shared vulnerabilities in several overlapping functional pathways. Most drug-resistant strains were more sensitive to tRNA synthetase knockdowns than the parental drug-sensitive strain, highlighting the potential of tRNA synthetases as high-value drug targets. Additionally, the rifampicin-resistant mutant RpoB(S450L) had increased sensitivity to the dysregulation of sulphur metabolism due to transcriptional dysregulation, although this vulnerability did not translate to all rpoB genotypes. Collateral vulnerabilities select for non-synonymous potentially adaptive mutations in metabolic pathways, reflecting a novel route of compensatory evolution. Combined, our findings highlight the power of functional genomics in pinpointing highly vulnerable drug targets across drug-resistant strains