Development of inducible genetic systems to finely tune efficacy of InhA-targeting anti-tuberculous drugs
- 1Aix Marseille Univ, CNRS, LISM, IM2B, Marseille, France
Abstract
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading global health threat, especially due to multidrug-resistant strains. The first-line drug isoniazid (INH) and second-line drugs ethionamide (ETH) and prothionamide (PTH), are prodrugs that target the essential enoyl-ACP reductase InhA, a key enzyme involved in mycolic acid biosynthesis. Resistance to these drugs primarily arises from genetic mutations that inactivate the enzymatic function of their respective bioactivating enzymes. While the recent development of chemical boosters has shown promising results in restoring drug susceptibility in these mutants, genetic tools to dynamically modulate these pathways and fine-tune resistance could be extremely valuable to develop the next-generation of anti-InhA drugs. In this context, we leverage CRISPR interference (CRISPRi) to develop inducible and reversible genetic systems to fine-tune antibiotic susceptibility in Mycobacterium smegmatis (Msmeg) and Mtb. By using our CRISPRi-based approach we first generated an inducible inhA hypomorph, an essential target required for mycolic acid production and bacterial growth, therefore confirming that our method is fully functional. Then, we developed genetic tools to selectively target katG, ethA, and the transcriptional repressor ethR, in order to generate conditional hypomorphs with altered antibiotic susceptibility profiles. Antibiotic susceptibility assays confirmed that the conditional hypomorphs selectively displayed high-level resistance or hypervulnerability to INH or ETH upon CRISPRi induction. Altogether, this work establishes CRISPRi as a powerful genetic platform for selectively fine-tuning mycobacterial drug susceptibility, with potential applications in developing next-generation anti-TB therapies.
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BibTeX
@misc{ramos2026development,
title = {{Development of inducible genetic systems to finely tune efficacy of InhA-targeting anti-tuberculous drugs}},
author = {E. Sepulveda Ramos and M. S. Deschutter and V. Point and T. Brunet and J. Laudouze and W. Achache and C. Crauste and S. Canaan and G. Gago and M. Maziero and P. SANTUCCI},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.25.754513},
url = {https://www.biorxiv.org/content/10.64898/2026.09.25.754513v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Development of inducible genetic systems to finely tune efficacy of InhA-targeting anti-tuberculous drugs
AU - E. Sepulveda Ramos
AU - M. S. Deschutter
AU - V. Point
AU - T. Brunet
AU - J. Laudouze
AU - W. Achache
AU - C. Crauste
AU - S. Canaan
AU - G. Gago
AU - M. Maziero
AU - P. SANTUCCI
PY - 2026
DA - 2026/09/25
PB - bioRxiv
DO - 10.64898/2026.09.25.754513
UR - https://www.biorxiv.org/content/10.64898/2026.09.25.754513v1
AB - Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading global health threat, especially due to multidrug-resistant strains. The first-line drug isoniazid (INH) and second-line drugs ethionamide (ETH) and prothionamide (PTH), are prodrugs that target the essential enoyl-ACP reductase InhA, a key enzyme involved in mycolic acid biosynthesis. Resistance to these drugs primarily arises from genetic mutations that inactivate the enzymatic function of their respective bioactivating enzymes. While the recent development of chemical boosters has shown promising results in restoring drug susceptibility in these mutants, genetic tools to dynamically modulate these pathways and fine-tune resistance could be extremely valuable to develop the next-generation of anti-InhA drugs. In this context, we leverage CRISPR interference (CRISPRi) to develop inducible and reversible genetic systems to fine-tune antibiotic susceptibility in Mycobacterium smegmatis (Msmeg) and Mtb. By using our CRISPRi-based approach we first generated an inducible inhA hypomorph, an essential target required for mycolic acid production and bacterial growth, therefore confirming that our method is fully functional. Then, we developed genetic tools to selectively target katG, ethA, and the transcriptional repressor ethR, in order to generate conditional hypomorphs with altered antibiotic susceptibility profiles. Antibiotic susceptibility assays confirmed that the conditional hypomorphs selectively displayed high-level resistance or hypervulnerability to INH or ETH upon CRISPRi induction. Altogether, this work establishes CRISPRi as a powerful genetic platform for selectively fine-tuning mycobacterial drug susceptibility, with potential applications in developing next-generation anti-TB therapies.
ER -