Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.
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This work was funded by grants from the US National Institutes of Health (R01 GM081875 and GM106239 to K.H.G.; R01 GM103383 to K.W.L.; R01 GM096164 to O.D.W.), Cancer Prevention and Research Institute of Texas (RP130312), Defense Advanced Research Projects Agency (Living Foundries HR0011-12-C-0068 to B. Chow (University of Pennsylvania), supporting S.G.) and the Robert A. Welch Foundation (I-1424 to K.H.G.). K.H.G. is the Virginia Lazenby O'Hara Chair in Biochemistry and W.W. Caruth Scholar in Biomedical Research. A.R. was supported by a National Science Foundation Graduate Research Fellowship. We thank S.L. McKnight and P.R. Potts (both at UT Southwestern Medical Center) for generously providing constructs.
L.B.M.M. and K.H.G. have filed US Patent Application PCT/US2012/065493 covering the method described in this paper.
Supplementary Results, Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Notes 1 and 3. (PDF 2701 kb)
MATLAB code for kinetic model (DOC 28 kb)
Z-stack of 70% epiboly embryo showing mosaic expression of mCherry after illumination with blue light. (AVI 20581 kb)
Z-stack of 70% epiboly embryo showing no expression of mCherry under dark conditions. (AVI 24709 kb)
Localization of fluorescent mCherry in the heart of a developing zebrafish embryo at 24 h.p.f. (AVI 399 kb)
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Motta-Mena, L., Reade, A., Mallory, M. et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat Chem Biol 10, 196–202 (2014). https://doi.org/10.1038/nchembio.1430
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