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

Authors

Daniel Raj D, Anand Kumar Maurya, Jitendra Singh, Mukesh Kumar G, Anirban Ramani, Alkesh Kumar Khurana, Shashank Purwar, Debasis Biswas

Published in

Maedica. Volume 21. Issue 2. Pages 495-503.

Abstract

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.
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.
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.
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.

PMID:
42416743
Bibliographic data and abstract were imported from PubMed on 08 Jul 2026.

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