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A small and highly sensitive red/far-red optogenetic switch for applications in mammals

Abstract

Optogenetic technologies have transformed our ability to precisely control biological processes in time and space. Yet, current eukaryotic optogenetic systems are limited by large or complex optogenetic modules, long illumination times, low tissue penetration or slow activation and deactivation kinetics. Here, we report a red/far-red light-mediated and miniaturized Δphytochrome A (ΔPhyA)-based photoswitch (REDMAP) system based on the plant photoreceptor PhyA, which rapidly binds the shuttle protein far-red elongated hypocotyl 1 (FHY1) under illumination with 660-nm light with dissociation occurring at 730 nm. We demonstrate multiple applications of REDMAP, including dynamic on/off control of the endogenous Ras/Erk mitogen-activated protein kinase (MAPK) cascade and control of epigenetic remodeling using a REDMAP-mediated CRISPR–nuclease-deactivated Cas9 (CRISPR–dCas9) (REDMAPcas) system in mice. We also demonstrate the utility of REDMAP tools for in vivo applications by activating the expression of transgenes delivered by adeno-associated viruses (AAVs) or incorporated into cells in microcapsules implanted into mice, rats and rabbits illuminated by light-emitting diodes (LEDs). Further, we controlled glucose homeostasis in type 1 diabetic (T1D) mice and rats using REDMAP to trigger insulin expression. REDMAP is a compact and sensitive tool for the precise spatiotemporal control of biological activities in animals with applications in basic biology and potentially therapy.

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Fig. 1: Construction and optimization of the REDMAP system.
Fig. 2: Characterization of the REDMAP system performance.
Fig. 3: REDMAP-mediated dynamic control of Ras/Erk MAPK signaling.
Fig. 4: REDMAP-controlled CRISPR–dCas9 system (REDMAPcas) for epigenome engineering.
Fig. 5: REDMAP-mediated transgene expression in mice.
Fig. 6: REDMAP-mediated insulin production to control glucose homeostasis in T1D mice and rats.

Data availability

All data associated with this study are present in the paper or the Supplementary Information. All genetic components related to this paper are available with a material transfer agreement and can be requested from H.Y. (hfye@bio.ecnu.edu.cn). Source data are provided with this paper.

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Acknowledgements

This work was financially supported by grants from the National Key R&D Program of China, Synthetic Biology Research (no. 2019YFA0904500), the National Natural Science Foundation of China (NSFC; no. 31971346, no.31861143016), the Science and Technology Commission of Shanghai Municipality (no. 18JC1411000), the Thousand Youth Talents Plan of China and the Fundamental Research Funds for the Central Universities to H.Y. This work was also partially supported by NSFC (no. 31901023 to N.G.). W.W. was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy, EXC-2189, project ID 390939984. We also thank the ECNU Multifunctional Platform for Innovation (011) for supporting the mouse experiments and the Instruments Sharing Platform of the School of Life Sciences, ECNU.

Author information

Authors and Affiliations

Authors

Contributions

H.Y. conceived the project. H.Y., Y.Z. and D.K. designed the experiment. Y.Z., D.K., X. Wang, X. Wu and G.Y. performed the experimental work. H.Y., Y.Z., N.G. and W.W. analyzed the results and wrote the manuscript. All authors edited and approved the manuscript.

Corresponding author

Correspondence to Haifeng Ye.

Ethics declarations

Competing interests

H.Y., Y.Z. and D.K. are inventors of patent applications (Chinese patent application number 202010443080.3) submitted by ECNU that cover the new optogenetic REDMAP system. All other authors declare that they have no competing interests.

Additional information

Peer review information Nature Biotechnology thanks Zhen Gu and Guoping Feng for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Characterization of the performance of the REDMAP system.

a, SEAP production of HEK293 cells transfected with REDMAP-encoding plasmids. HEK293 cells transfected with REDMAP-encoding plasmids were illuminated with red light (660 nm, 1 mW/cm2) for 1 min. The control cells were transfected with different expression vectors to yield different combinatorial configurations for the presence (+) or absence (-) of ΔPhyA-Gal4, FHY1-VP64 and SEAP reporter. ***P < 0.0001. b, Constitutive gene expression of human cells exposed to far-red, red and blue light, respectively. HEK293 cells were transfected with 100 ng pSEAP2-Control, followed by illumination (1 mW/cm2, 660 nm, 730 nm, or 465 nm) for 1 h. ***P < 0.0001. c, Viability of human cells exposed to far-red, red and blue light, respectively. HEK293 cells bearing the REDMAP system were illuminated with different wavelengths of light (1 mW/cm2, 660 nm, 730 nm, or 465 nm) for 1 h. ***P < 0.0001. d, REDMAP–mediated transgene expression in different mammalian cell lines. Different mammalian cell lines were co-transfected with REDMAP-encoding plasmids. Twenty-four hours after transfection, cells were illuminated with red light (1 mW/cm2) for 1 min, and SEAP expression in the culture supernatant was scored 24 h after illumination. Data are expressed as means ± SD; n = 3 independent experiments. P values were calculated by two-tailed unpaired t test. N.S., not significant. Detailed description of genetic components and transfection mixtures are provided in Supplementary Table 1 and 5.

Source data

Extended Data Fig. 2 Assessment of the compatibility of phycocyanobilin (PCB) with HEK-293 cells and Mice.

a, Viability of HEK293 cells after exposure to various concentrations of PCB. HEK293 cells were seeded into each well of a 96-well plate and cultivated in medium containing the indicated PCB concentrations (0–100 µM) for 24 h before cell viability was evaluated with CCK-8 assays. b, Viability of HEK293 cells after transfection with various amounts of PCB-producer plasmid pDQ55 (0–200 ng). HEK293 cells were seeded into each well of a 96-well plate and transfected with the indicated amounts of PCB-producer plasmid pDQ55. Twenty-four hours later, cell viability was evaluated with CCK-8 assays. c–m, Compatibility of PCB with mice. Mice were intraperitoneally injected with PCB (5 mg/kg/day) or PBS (once daily) for three weeks. c, Body weight was monitored every three days for a total of three weeks. d, Hematoxylin and eosin (H&E) staining for the liver tissues in mice treated with 5 mg/kg/day via intraperitoneal injection. H&E staining, × 200 magnification. Scale bars, 100 μm. e–m, On day 21, mouse blood was collected for the blood biochemical analysis, liver function and hepatic function analysis. (e) Counts for total white blood cells (WBC), (f) red blood cells (RBC), (g) hemoglobin (HGB), (h) platelet (PLT). i, j, Kidney function analysis. (i) blood urea nitrogen (BUN), (j) creatinine (CRE). k–m Hepatic function analysis. (k) aspartate aminotransferase (AST), (l) alanine aminotransferase (ALT), (m) albumin/globulin ratio (A/G). Data in a, b, are expressed as means ± SD; n = 3 independent experiments. Data in c, e–m are expressed as means ± SEM (n = 6 mice). P values were calculated by two-tailed unpaired t test. N.S., not significant. Detailed description of genetic components and transfection mixtures are provided in Supplementary Table 1 and 5.

Source data

Extended Data Fig. 3 Quantification of REDMAP activity with different amounts of the PCB-producer plasmid (pDQ55).

HEK293 cells were co-transfected with pYZ181, pDQ16, pDL6, x ng pDQ55 (PhCMV-PcyA-P2A-HO-P2A-Fd-P2A-FNR-pA) and (200-x) ng pcDNA3.1(+). Twenty-four hours after transfection, the cells were illuminated with red light (1 mW/cm2) for 1 min and SEAP expression in the culture supernatant was quantified 24 h after illumination. ***P < 0.0001 for all comparisons. Data are expressed as means ± SD; n = 3 independent experiments. P values were calculated by two-tailed unpaired t test. N.S., not significant. Detailed description of genetic components and transfection mixtures are provided in Supplementary Table 1 and 5.

Source data

Extended Data Fig. 4 Characterization of the performance of the REDMAP system.

a–g, Light-induced SEAP expression from REDMAP-engineered cells preincubated with PCB. a, Experimental procedure for evaluating the binding properties of PCB. HEK293 cells were transfected with REDMAP-encoding plasmids, and PCB was added into the culture medium at 24 h after transfection. After incubation for 30 min, the culture medium was replaced with fresh medium without PCB and the transfected cells were divided into three groups, which were cultivated for different times (6, 12, or 24 h) before illuminating with red light. b–d, Light-induced SEAP production from REDMAP-engineered cells preincubated with PCB. In bd, ***P < 0.0001. e, Experimental procedure for evaluating the binding properties of PCB. HEK293 cells transfected with REDMAP-encoding plasmids were incubated with PCB for 30 min and then illuminated with red light. The illuminated cells were divided into two groups. One group of cells were cultivated with fresh medium supplemented with PCB, while the other group without PCB. f, g, Light-induced SEAP production from REDMAP-engineered cells incubated with or without PCB after 1 min of illumination. In f, g, ***P < 0.0001. h, Fluorescence micrograph profiling of EGFP expression kinetics mediated by the REDMAP system. Scale bar, 100 μm. Representative data from three independent experiments. i, Comparison of REDMAP with other red/far-red light-inducible transgene expression systems. j, Illumination time-dependent REDMAP-mediated OFF kinetics of transgene expression. HEK293 cells were illuminated with red light. One hour after red light illumination, cells were exposed to far-red light (1 mW/cm2) for different time periods (0 to 10 min). ***P < 0.0001 for all groups versus 660 nm. k, Illumination intensity-dependent REDMAP-mediated OFF kinetics of transgene expression. HEK293 cells were illuminated with red light. One hour after red light illumination, cells were exposed to far-red light of various light intensities (0 to 1 mW/cm2) for 1 min. ***P < 0.0001 for all groups versus 660 nm. Data are expressed as means ± SD; n = 3 independent experiments. P values were calculated by two-tailed unpaired t test. N.S., not significant. Detailed description of genetic components and transfection mixtures are provided in Supplementary Table 1 and 5.

Source data

Extended Data Fig. 5 Control experiments for main Fig. 3b.

HEK293 cells transfected with REDMAPSOS-Ras (ΔPhyA-CAAX and FHY1-SOScat) were illuminated with red light (1 mW/cm2) for 1 min. Ten minutes after illumination, the cells were harvested and probed for phospho-Erk1/2, total Erk1/2 (tErk) and GAPDH. Negative control cells were only transfected with pcDNA3.1(+). Representative data from two independent experiments. Detailed description of genetic components and transfection mixtures are provided in Supplementary Table 1 and 5.

Source data

Extended Data Fig. 6 A comparison of endogenous gene activation with the REDMAPcas system and the far-red light-activated CRISPR-dCas9 (FACE) system.

a, Experimental procedure for light-controlled transcriptional activation of endogenous gene in HEK293 cells. b, Light-controlled transcriptional activation of endogenous gene ASCL1 in HEK293 cells mediated by REDMAPcas and FACE. HEK293 cells were transfected with REDMAPcas [pSZ69 (PhCMV-dCas9-pA), pYZ181, pDQ16, pDQ100 (P5×UAS-MS2-p65-HSF1-pA, P5×UAS, 5×UAS-PhCMVmin)] or FACE [pSZ69, pGY102 (PFRL1b-FGTA4-pA; PFRL1b, (whiG)2-PhCMVmin3G; FGTA4, MS2-P65-HSF1), pWS189, pGY32] together with two gRNAs targeting the ASCL1 gene [pSZ83 (PU6-sgRNA1ASCL1-pA) and pSZ84 (PU6-sgRNA2ASCL1-pA)], and were illuminated with red light (1 mW/cm2) or far-red light (1 mW/cm2) for 1 min. ***P < 0.0001. Data are expressed as means ± SD; n = 3 independent experiments. P values were calculated by two-tailed unpaired t test. N.S., not significant. Detailed description of genetic components and transfection mixtures are provided in Supplementary Table 1 and 5.

Source data

Extended Data Fig. 7 Optimization and characterization of the REDMAP system in mice.

a, Schematic showing the time schedule and experimental procedure for REDMAP-mediated transgene expression in mice. b, REDMAP-mediated transgene expression in mice. Mice were hydrodynamically injected with pDQ59 (PHYA-GAL4::FHY1-VP64), pYZ360 (5×UAS-PhCMVmin) and intraperitoneally injected with PCB (3.65 mg/kg) before illumination. **P = 0.0014. c, Co-delivery of the PCB-producer plasmid in mice. Mice hydrodynamically injected with pDQ59, pYZ360, and a PCB-producer plasmid pDQ55 were illuminated with red light. ***P = 0.0006. d, An iterative REDMAP-mediated transgene expression in mice. Mice were hydrodynamically injected with pDQ59, pYZ450 (5×UAS-PTATA), and pDQ55, and illuminated with red light. **P = 0.0018. Data in bd are expressed as means ± SEM (n = 4 mice). P values were calculated by two-tailed unpaired t test. N.S., not significant. e, PCB concentration-dependent REDMAP-mediated luciferase expression kinetics in mice. Mice were hydrodynamically injected with pDQ59, pYZ450. Eight hours after injection, mice were intraperitoneally injected with various amounts of PCB (0–5 mg/kg) before illumination. ***P = 0.0001, ***P = 0.0007, ***P < 0.0001, ***P < 0.0001, left to right. Data are expressed as means ± SEM (n = 5 mice). P values were calculated by two-tailed unpaired t test. f, The activation and deactivation performance of the REDMAP system in mice. Mice were hydrodynamically injected with pDQ59, pYZ450, and pDQ55, and mice were then divided into three groups. The control group was kept in the dark. Eight hours after injection, the mice in group A and B were illuminated with red light to activate the REDMAP system. Mice in group B were immediately illuminated with far-red light to deactivate REDMAP system. ***P < 0.0001 for both comparisons. Data are expressed as means ± SEM (n = 6 mice). P values were calculated by two-tailed unpaired t test. Detailed images of the bioluminescence signal are provided in Supplementary Fig. 3 and detailed description of genetic components and transfection mixtures are provided in Supplementary Table 1 and 5.

Source data

Extended Data Fig. 8 Serum inflammatory cytokine and whole blood analysis of AAV REDMAP mice.

Mice were injected via tail vein with AAV encoding the REDMAP system. Control mice were injected with PBS. Twelve weeks after injection, mouse blood was collected for the quantification of (a) serum IL-6, (b) serum IFN-γ, and (c) serum TNF-α production by ELISA. d Counts for total white blood cells (WBC), blood lymphocytes, and blood monocytes, in mice. Data are expressed as means ± SEM (n = 6 mice). P values were calculated by two-tailed unpaired t test. N.S., not significant.

Source data

Extended Data Fig. 9 REDMAP-mediated SEAP production in animals.

a, HEK-293 cells transfected with REDMAP system were microencapsulated into coherent alginate-poly- (L-lysine)-alginate beads (400 μm; 200 cells per capsule) and subcutaneously implanted into mice, rats, and rabbits. The animals were illuminated from the top with red light (20 mW/cm2) and blood SEAP was quantified 48 h after the first illumination. bd, Light-induced SEAP expression in mice (b), rats (c), and rabbits (d). b, Mice implanted with 2.5×106 microencapsulated transgenetic HEK-293 cells and illuminated daily for 1 min. ***P < 0.0001. c, Rats implanted with 1.2×107 microencapsulated transgenetic HEK-293 cells and illuminated twice daily for 5 min. ***P < 0.0001. d, Rabbits implanted with 3×107 microencapsulated HEK-293 transgenic cells and illuminated twice daily for 5 min. Control animals were implanted with microencapsulated wild-type HEK293 cells. ***P < 0.0001. Data are presented as means ± SEM (n = 5 animals). P values were calculated by two-tailed unpaired t test. N.S., not significant.

Source data

Extended Data Fig. 10 Characterization of the REDMAP-mediated SEAP and insulin expression stable cell lines.

The HEKREDMAP-SEAP-P2A-mINS cell line, transgenic for red light-inducible SEAP expression, was constructed by co-transfecting HEK293 cells with pYZ474 (ITR-P5×UAS*-SEAP-P2A-insulin-pA::PmPGK-ZeoR-pA-ITR) and pYZ484 (ITR-PhCMV-ΔPhyA-Gal4-P2A-FHY1-VP64-pA::PhCMV-PcyA-P2A-HO-P2A-Fd-P2A-FNR-pA::PmPGK-Puro-pA-ITR), and selected with puromycin (1 μg/mL) and zeocin (100 μg/mL) for two weeks. Twenty randomly selected cell clones were profiled for their light-inducible SEAP production performance by cultivating them for 24 h after illumination with red light (1 mW/cm2) for 1 min.

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Zhou, Y., Kong, D., Wang, X. et al. A small and highly sensitive red/far-red optogenetic switch for applications in mammals. Nat Biotechnol 40, 262–272 (2022). https://doi.org/10.1038/s41587-021-01036-w

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