A photoswitchable HaloTag for spatiotemporal control of fluorescence in living cells

Photosensitive fluorophores, which emission can be controlled using light, are essential for advanced biological imaging, enabling precise spatiotemporal tracking of molecular features, and facilitating super-resolution microscopy techniques. While irreversibly photoactivatable fluorophores are well established, reversible reporters which can be re-activated multiple times remain scarce, and only few have been applied in living cells using generalizable protein labelling methods. To address these limitations, we introduce chemigenetic photoswitchable fluorophores, leveraging the self-labelling HaloTag protein with fluorogenic rhodamine dye ligands. By incorporating a light-responsive protein domain into HaloTag, we engineer a tunable, photoswitchable HaloTag (psHaloTag), which can reversibly modulate the fluorescence of a bound dye-ligand via a light-induced conformational change. Our best performing psHaloTag variants show high performance in vitro and in living cells, with large, reversible, far-red fluorescence turn-on upon 450 nm illumination across various biomolecular targets. Together, this work establishes the chemigenetic approach as a versatile platform for the design of photoswitchable reporters, tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging.