AlphaFold3-guided engineering of light-controlled endonuclease

We developed MagMboI-plus, a photoactivatable restriction enzyme optimized for light-inducible genome editing. Based on the original MagMboI construct, MagMboI-plus was redesigned using AlphaFold3 to improve DNA binding and cleavage activity. Structural modeling guided the identification of a more stable split-protein configuration targeting the GATC recognition site. In Saccharomyces cerevisiae, MagMboI-plus exhibited enhanced DNA cleavage and triggered more extensive genome rearrangements upon blue light exposure. This study highlights the utility of structure prediction for engineering synthetic enzymes and supports MagMboI-plus as a precise tool for light-controlled top-down genome engineering.