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While powerful, the LOV-insertion strategy is still limited by the difficulty of finding a suitable insertion site to mediate a strong photoswitchable effect.

Since these initial studies, similar approaches have been used to develop light-controlled nanobodies and monobodies against multiple target proteins 5, 6, light- and temperature-controlled systems for regulating Cas9 7, 8, and light-based regulation of mammalian pyruvate kinase activity 9. Indeed, studies found that the AsLOV2 domain could be used to allosterically control proteins of interest, provided that a suitable insertion site could be found 2, 3, 4. Upon illumination, a conformational change in the light-sensitive domain can then propagate to the attached protein, potentially altering its functional state. One promising technique for engineering photoswitchable protein variants involves the internal insertion of a light-sensitive domain such as the LOV2 domain from Avena sativa Phototropin 1 (AsLOV2). Without detailed knowledge of a target protein’s structure and mechanism of activation, it remains challenging to incorporate photoswitchable domains in a manner that enables light-gated regulation of the target protein’s activity. One limitation faced by the field is the development of general strategies to obtain light-switchable variants of any protein of interest. Yet despite their widespread adoption, light-switchable variants are still only available for a limited subset of highly studied proteins and pathways. Optogenetic tools are increasingly used to interrogate a wide range of biological processes, joining small molecule inhibitors and agonists as standard perturbative tools available to cell and developmental biologists 1. Our approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited. We show that light-switchable function generalizes to analogous insertion sites in two additional Cys6Zn2 and C2H2 zinc finger domains, providing a starting point for optogenetic regulation of a broad class of transcription factors. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. We demonstrate the approach’s utility using the Gal4-VP64 transcription factor as a model system. The approach is based on insertion of the AsLOV2 photoswitchable domain at all possible positions in a candidate protein of interest, introduction of the library into mammalian cells, and light/dark selection for variants with photoswitchable activity. Here, we adapt strategies for protein domain insertion and mammalian-cell expression to generate and screen a library of candidate optogenetic tools directly in mammalian cells. Yet the development of new light-switchable protein variants remains challenging, and the field still lacks general approaches to engineering or discovering protein variants with light-switchable biological functions.

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