Rational design of site-specific artificial metallonucleases for therapeutic applications

• Introduces the fundamental mechanisms and strategies employed in the design of site-specific artificial metallonucleases (ArMNs). • Reviews significant examples of ArMNs developed over the past 20 years, with emphasis on substrate recognition and cleavage precision. • Highlights a wide range of therapeutic applications, including gene editing, antiviral, and anticancer interventions. • Discusses innovative approaches to enhance the performance of ArMNs for future clinical and biotechnological use. Artificial metallonucleases (ArMNs) are synthetic nucleases that utilise metals as the catalytic centre for cleaving nucleic acids, with the metal component present in the form of ions, complexes or nanoparticles. ArMNs have been developed as therapeutics for a range of diseases, including genetic disorders, cancer and infectious diseases. Achieving high site-specificity in ArMNs is crucial for their effective application in targeted therapeutic interventions. By employing advanced methodologies, these engineered nucleases can induce targeted modifications in the genome, thereby advancing genetic research, improving disease treatment and fostering biotechnological innovation. This review examines recent advancements over the past 20 years in the design and synthesis of site-specific ArMNs. Illustrated examples are provided to elucidate the underlying principles of their site-specific activity and to highlight their prospective applications. Additionally, strategies for overcoming challenges in enhancing the selectivity of these nucleases are discussed, and anticipated future research directions in this dynamic field are outlined.