Light-switchable transcription factors obtained by direct screening in mammalian cells

Liyuan Zhu, Harold M. McNamara and Jared E. Toettcher ()
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Liyuan Zhu: Princeton University
Harold M. McNamara: Princeton University
Jared E. Toettcher: Princeton University

Nature Communications, 2023, vol. 14, issue 1, 1-16

Abstract: Abstract Optogenetic tools can provide fine spatial and temporal control over many biological processes. 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. 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. 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. We demonstrate the approach’s utility using the Gal4-VP64 transcription factor as a model system. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. 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 approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited.

Date: 2023
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DOI: 10.1038/s41467-023-38993-6

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