Abstract
Red light penetrates deep into mammalian tissues and has low phototoxicity, but few optogenetic tools that use red light have been developed. Here we present MagRed, a red light–activatable photoswitch that consists of a red light–absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin and a photo-state-specific binder that we developed using Affibody library selection. Red light illumination triggers the binding of the two components of MagRed and the assembly of split-proteins fused to them. Using MagRed, we developed a red light–activatable Cre recombinase, which enables light-activatable DNA recombination deep in mammalian tissues. We also created red light–inducible transcriptional regulators based on CRISPR–Cas9 that enable an up to 378-fold activation (average, 135-fold induction) of multiple endogenous target genes. MagRed will facilitate optogenetic applications deep in mammalian organisms in a variety of biological research areas.
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Data availability
The data supporting the findings of this study are available within the article and its Supplementary Information. The source data for the main figures and extended data figures are provided as Source Data files. A crystal structure of Affibody (Protein Data Bank accession code 2M5A) was used to depict a schematic representation of a binding partner candidate in Fig. 1b. The read counts for all screening data are available on the DDBJ Sequence Read Archive, accession numbers DRR243933 and DRR243934. Source data are provided with this paper.
Code availability
The code for analysis of the read counts for all screening data has been deposited on GitHub (https://github.com/Kazushi40/NGS_analysis).
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Acknowledgements
We thank Y. Aono for support in the analysis of biochemical experiments. This work was supported by a CREST grant (JPMJCR1653) from the Japan Science and Technology Agency (to R.N., M.Y. and M.S.); a project grant from the Kanagawa Institute of Industrial Science and Technology; and the UTEC-UTokyo FSI Research Grant Program to M.S. Y. Kuwasaki was supported by KAKENHI-PROJECT-20J14492 for the Japan Society for the Promotion of Science (JSPS) Doctoral Course Students Research Fellow. K.S. was supported by Grant-in-Aid 18J01772 for the JSPS Research Fellow.
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M.S. conceived the project and provided supervision. G.Y. developed the synthetic binder, with support from Y.S., and performed preliminary tetR-tetO experiments. R.N., K.M., K.F. and Y. Kuwasaki measured absorption spectra of DrBphP and RpBphP1. Y. Kuwasaki, S.Y. and K.M. performed biochemical study using QCM. Y. Kuwasaki and M.N. performed split-fluc reassembly assays. Y. Kakihara and Y. Kuwasaki performed experiments of tetR-tetO. Y. Kuwasaki, T.O. and Y. Kakihara performed experiments of Red-CPTS, with help from T.N. Y. Kuwasaki, M.N., K.S., R.B. and M.Y. performed experiments of RedPA-Cre. Y. Kuwasaki, K.S. and M.S. wrote the manuscript and prepared the figures. R.B. and M.Y. edited the manuscript. All authors checked and approved the final manuscript.
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Extended data
Extended Data Fig. 1 Effect of additional BV supplementation on the three different CPTS designs.
(a-c) Mean bioluminescence intensities (from three independent biological samples) of CPTS designs based on RpBphP1-PpsR2 (a), RpBphP1-QPAS1 (b), and MagRed (c) were plotted. The designs of each configuration (#1-8) were shown in Fig. 3b, c. P values are indicated above the bars. (N.S., not significant P > 0.05; **P < 0.01; BV minus vs. plus using two-tailed Wilcoxon matched-pairs signed rank test).
Extended Data Fig. 2 Red-CPTS can be actively switched off using 800-nm light illumination.
(a-d) After the pre-activation of Red-CPTS by the 660-nm illumination for 12 h, the cells were further incubated under the 660-nm illumination condition (a) or switched to the dark condition (b) or the 800-nm illumination condition (c). As a control, the sample was incubated under the dark condition throughout the experiment (d). ASCL1 mRNA levels were measured at 0 h, 0.75 h, 1.5 h and 3.0 h as shown in the figure. Box plots show the median (center line), first and third quartiles (box edges), 1× the SD (whiskers), and individual data points. (n = 6 biologically independent samples, mean ± s.d.).
Extended Data Fig. 3 Comparison of Red-CPTS and RpBphP1-PspR2/QPAS1-based CPTS at different illumination intensities.
(a-c) Fluc reporter gene activation by Red-CPTS (a), RpBphP1-PpsR2-based CPTS (b), and RpBphP1-QPAS1-based CPTS (c) at various illumination intensities. RpBphP1-PpsR2/QPAS1-based CPTS has configuration #3 shown in Fig. 3b, c. Experimental conditions are same as those in Fig. 3c, d except for the illumination intensities. Ratios of the mean bioluminescence intensity under the red light condition (red bar) to that under the dark condition (gray bar) are depicted above the bars. Bar data are shown as the mean ± s.d. from four biological replicates. Dots represent individual data points. (d) Left: Comparison of the Light/Dark contrasts between Red-CPTS and RpBphP1-PpsR2/QPAS1-based CPTS at different illumination intensities. Right: The cropped data with the mean Light/Dark contrast of 0 to 3.
Extended Data Fig. 4 Dependence of Red-CPTS activity on the duration of ON time of the red light illumination.
(a-c) Fluc reporter gene activation by Red-CPTS (a), RpBphP1-PpsR2 (b), and RpBphP1-QPAS1 (c) with various durations of ON time of the red light illumination. RpBphP1-PpsR2/QPAS1-based CPTS has configuration #3 shown in Fig. 3b, c. Experimental conditions are the same as those in Fig. 3c, d except for the illumination cycle. Ratios of the mean bioluminescence intensity under the red light condition (red bar) to that under the dark condition (gray bar) are depicted above the bars. Bar data are shown as the mean ± s.d. from four biological replicates. Dots represent individual data points. (d) Left: Comparison of the Light/Dark contrasts between Red-CPTS and RpBphP1-PpsR2/QPAS1-based CPTS at various durations of ON time of the red light illumination. Right: The cropped data with the mean Light/Dark contrast of 0 to 3.
Extended Data Fig. 5 Dependence of Red-CPTS activity on the duration of OFF time of the red light illumination.
(a-c) Fluc reporter gene activation by Red-CPTS (a), RpBphP1-PpsR2 (b), and RpBphP1-QPAS1 (c) with various durations of OFF time of the red light illumination. RpBphP1-PpsR2/QPAS1-based CPTS has configuration #3 shown in Fig. 3b, c. Experimental conditions were the same as those in Fig. 3c, d except for the illumination cycle. Ratios of the mean bioluminescence intensity under the red light condition (red bar) to that under the dark condition (gray bar) are depicted above the bars. Bar data are shown as the mean ± s.d. from four biological replicates. Dots represent individual data points. (d) Left: Comparison of the Light/Dark contrasts between Red-CPTS and RpBphP1-PpsR2/QPAS1-based CPTS at various durations of OFF time of the red light illumination. Right: The cropped data with the mean Light/Dark contrast of 0 to 3.
Extended Data Fig. 6 Comparison of RedPA-Cre with the existing red light-responsive recombinase systems.
For the BV/PCB (+) conditions, HEK 293T cells were plated at 2.0 × 104 cells per well in a 96-well black-wall plate in the presence of 25 μM BV (for RedPA-Cre and FISC system) and 20 μM PCB (for CreLite and L-SCRaMbLE), respectively. For the BV/PCB(−) conditions, the procedures for plating were identical to those described above except for the chromophore supplementation. Plasmid amount used for each experiment is described below the graph. Especially, because a previous study has revealed that FISC system shows the highest recombination efficiency when Cre N-fragment (pXY169), Cre C-fragment (pXY177) and red light-responsive activator (pXY137) were transfected at 1:1:10 ratio, we additionally tested this transfection condition for FISC system. Following experimental procedures are the same as those in Fig. 5c, d. Ratios of the mean bioluminescence intensity under the red light condition (red bar) to that under the dark condition (gray bar) are depicted above the bars. Bar data are shown as the mean ± s.d. from four biological replicates. Dots represent individual data points. P values are indicated above the bars. (N.S., not significant P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; dark vs. light using two-tailed unpaired t-test).
Extended Data Fig. 7 RedPA-Cre activation with various red-light illumination conditions.
HEK 293T cells were transfected with plasmids encoding RedPA-Cre and the bioluminescence reporter without additional BV supplementation. Twenty-four hours after the transfection, the cells were illuminated with red light at different illumination durations and intensities. In the left panels showing the illumination conditions, gray corresponds to the dark condition and red with asterisk corresponds to the red light condition with denoted light intensities. Ratios of the mean bioluminescence intensity under the red light condition (red bar) to that under the dark condition (gray bar) are depicted above the bars. Bar data are shown as the mean ± s.d. from four biological replicates. Dots represent individual data points. P values are indicated above the bars. (****P < 0.0001; one-way ANOVA with multiple comparisons).
Extended Data Fig. 8 RedPA-Cre under dark, 800-nm illumination, and 660-nm illumination conditions.
DNA recombination activities of RedPA-Cre were compared among the dark, the 800-nm illumination and the 660-nm illumination conditions. Experimental conditions are the same as those in Fig. 5c, d except for that the 800-nm samples are incubated under 800-nm pulsed light (1 min ON and 4 min OFF) of 10 W m−2. Bar data are shown as the mean ± s.d. from four biological replicates. Dots represent individual data points. P values are indicated above the bars. (N.S., not significant P > 0.05; ****P < 0.0001; dark vs. 660 nm, dark vs. 800 nm and 800 nm vs. 660 nm using two-tailed unpaired t-test).
Extended Data Fig. 9 RedPA-Cre enables for DNA recombination reaction upon noninvasive red light illumination in living mice.
(a, b) ICR mice were transfected with a bioluminescent reporter plasmid together with pcDNA3.1 empty plasmid as a control (a) or with plasmid encoding RedPA-Cre of which configuration is NLS-DrBphP-CreC106-IRES-NLS-CreN104-Aff6_V18FΔN (b). Twenty-five hours after the transfection, bioluminescence images were obtained. No difference can be observed in the appearance between the mice maintained in the dark and the ones illuminated with red light at 660 nm for 16 h. The total bioluminescence intensities were shown in Fig. 5f (n = 3 mice per group).
Extended Data Fig. 10 In vivo gene activation by Red-CPTS upon noninvasive red light illumination.
(a, b) ICR mice were transfected with plasmids encoding Red-CPTS and luciferase reporter together with a plasmid encoding unrelated sgRNA as a negative control (a, Empty) or sgRNA targeting GAL4UAS (b, GAL4UAS). After the transfection, the mice were noninvasively illuminated at 660 nm or kept in the dark as shown in Supplementary Figure 28, and then bioluminescence imaging of the mice was performed. (c) Total bioluminescence intensities of the mice shown in a and b. Gray and red bars represent the mean ± s.d., and dots represent the total bioluminescence intensity of each mouse (n = 4 mice per group). (N.S. P > 0.05; ****P < 0.0001; using two-way ANOVA with multiple comparisons). (d) Red light-dependent endogenous gene activation by Red-CPTS with unrelated sgRNA as a negative control (Empty) or sgRNA targeting mouse ASCL1 (mASCL1) in vivo in living BALB/c mice. Data are represented as the relative mRNA level to the non-transfected negative control (n = 6 mice per group). Gray and red bars represent the mean ± s.d., and dots represent individual data points. No difference can be observed in the appearance between the mice maintained in the dark and the ones illuminated with red light at 660 nm for 16 h. P values are indicated above the bars. (N.S., not significant P > 0.05; ****P < 0.0001; using two-way ANOVA with multiple comparisons).
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Kuwasaki, Y., Suzuki, K., Yu, G. et al. A red light–responsive photoswitch for deep tissue optogenetics. Nat Biotechnol 40, 1672–1679 (2022). https://doi.org/10.1038/s41587-022-01351-w
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DOI: https://doi.org/10.1038/s41587-022-01351-w
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