CRISPR-CAS THERAPEUTICS AGAINST EXTENSIVELY DRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS: OPPORTUNITIES FOR PRECISION ANTIMICROBIAL INTERVENTION

Abstract

Extensively drug-resistant tuberculosis (XDR-TB) remains a critical global health threat, largely due to the toxicity, prolonged treatment durations, and severe dysbiosis associated with conventional broad-spectrum antibiotic regimens. To address this, we evaluated the in vitro and in vivo efficacy of CRISPR-Cas therapeutics targeting key XDR-TB resistance mutations, deploying distinct modalities including double-strand break (DSB) nucleases (Cas9, Cas12a) for targeted bacterial killing and base editors (CBE/ABE) for resistance allele correction, delivered via lipid nanoparticles (LNPs) or engineered bacteriophages (BxP-1). In vitro, DSB-targeting Cas9 and Cas12a achieved high bactericidal activity against rpoB (S450L) and katG (S315T) mutants, yielding 99.2% and 98.5% kill rates, respectively, while multiplexed Cas9 targeting both loci achieved near-complete clearance (99.9% kill). Conversely, base editors targeting gyrA (D94G) and rrs (A1401G) resensitized resistant strains without direct killing, successfully restoring moxifloxacin and amikacin MICs to 0.25 μg/mL and 1.0 μg/mL. In vivo, the multiplexed rpoB + katG Cas9 therapy demonstrated the highest efficacy with a 3.5 ± 0.2 Δ log₁₀ CFU reduction in lung burden, outperforming the standard XDR-TB small-molecule regimen (3.1 ± 0.3 Δ log₁₀ CFU), with single target DSB and base-editing therapies also yielding substantial reductions (1.4–2.8 Δ log₁₀ CFU). Crucially, all CRISPR-Cas modalities exhibited exceptional microbiome preservation, with Microbiome Sparing Indices ranging from 0.92 to 0.98, compared to a significantly depleted index of 0.45 for the standard antibiotic regimen. Ultimately, these findings demonstrate that CRISPR-Cas therapeutics, particularly multiplexed DSB approaches and targeted base editors, provide highly effective, mutation-specific clearance and resensitization of XDR-TB, representing a promising next-generation paradigm for treating drug-resistant tuberculosis with minimized host collateral damage.