Off-Target activity as a Translational Barrier in Programmable Gene-Editing Strategies for Nontuberculous Mycobacteria: Narrative Review.

OBJECTIVES

To review the clinical and translational implications of off-target activity associated with clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches in nontuberculous mycobacteria (NTM) and discuss current strategies aimed at specificity and safety.

MATERIALS AND

METHODS

The relevant published literature on the application of CRISPR-Cas systems, including Cas9, Cas12a and CRISPR interference (CRISPRi), in NTM research was reviewed. Particular attention was given to off-target mechanisms, mycobacteria-specific genomic challenges, computational predictions, experimental detection methods, high-fidelity nucleases and delivery optimisation approaches.

RESULTS

Nontuberculous mycobacteria infections often require prolonged treatment and are frequently associated with relapse and rising antimicrobial resistance, particularly in Mycobacterium abscessus infections. CRISPR-based technologies provide advantages in precision diagnostics, functional genomics and therapeutic development; however, high guanine-cytosine (GC) content, repetitive PE/PPE gene families, mismatch tolerance and unique DNA repair mechanisms contribute considerably to off-target effects. Emerging high-fidelity nucleases, guide RNA optimisation, artificial intelligence (AI)-assisted prediction platforms and alternative editing systems demonstrate considerable potential for improving editing specificity and translational safety.

CONCLUSIONS

Advances in nuclease engineering, computational modelling, delivery systems, and genome-wide validation approaches may improve therapeutic precision and diagnostic reliability. Addressing these challenges through interdisciplinary innovation will be essential for the future clinical integration of CRISPR-based antimycobacterial strategies.