REDMAP：A New Generation of Optogenetic Tools
REDMAP is a red/far-red light-mediated and miniaturized Δphytochrome A (ΔPhyA)-based photoswitch system, with rapid activation/deactivation kinetics, high tunability, small construct size, and spatiotemporally programmable expression characteristics.
Optogenetics has opened up exciting new opportunities for precision-guided medicine by using illumination with light as a trigger signal to achieve pinpoint spatiotemporal control of cellular activities1. Optogenetics-based technology promises the capability to achieve traceless, remotely controlled precision dosing of an enormous range of therapeutic outputs for precision medicine .
As an emerging discipline, our laboratory has focused on the development of new optogenetic tools for biomedical applications. In 2011, Dr. Haifeng Ye developed a blue light-responsive optogenetic switch to trigger insulin production to control blood glucose homeostasis in a diabetic mouse model2. However, the nonorthogonality of the genetic circuit and the poor tissue penetration of blue light limited its further application.
Later, in 2017, our group developed a far-red light-controlled transgene expression system based on BphS3, which offered strong transcriptional activation. The system was further combined with CRISPR/Cas9, CRISPR/dCas9 and Cre recombinase to achieve controllable epigenome engineering4, gene editing5 and DNA recombination6 both in vitro and in vivo. However, there were also some limitations with this optogenetic tool, which required continuous illumination for several hours to fully activate its function. Due to the complexity and relatively large genetic modules, it is difficult to package AAV vectors for gene therapy. Therefore, we aimed to develop a simple design that is small enough for easy packaging into AAV vectors for efficient in vivo delivery, as well as provide rapid activation/deactivation features in response to light.
To achieve this goal, we reviewed the extensive literature and decided to employ a red/far-red light-responsive photoreceptor (PhyA) from Arabidopsis thaliana to engineer a new optogenetic tool. The chromophore phycocyanobilin (PCB) covalently binds to PhyA, resulting in photochemically functional photoreceptors in response to red (λmax = 660 nm) or far-red light (λmax = 730 nm). Under 660 nm illumination, PhyA rapidly binds to the shuttle protein FHY1, while under 730 nm illumination, PhyA and FHY1 immediately dissociate from each other. Taking advantage of these features, we designed a red/far-red light-mediated photoswitch (REDMAP) for use in mammals (Fig. 1)7.
Initially, we attempted to fuse full-length PhyA to the yeast Gal4 DNA binding domain to form a hybrid DNA binding protein (PhyA-Gal4), and the PhyA interaction protein FHY1 was fused to the transcriptional activator VP64 to form a hybrid light-dependent transactivator (FHY1-VP64). Under illumination, FHY1-VP64 specifically binds to PhyA-Gal4, and the combined protein complex translocates into the nucleus, where it binds to its synthetic promoter (P5×UAS, 5×UAS-PhCMVmin) to initiate transcription of the downstream reporter gene. Unfortunately, we did not observe significant activation of transgene expression.
We speculated that the large size of full-length PhyA (1126 aa) causes steric hindrance during transcriptional activation. Subsequently, we referred back to the literature and tried to test different truncated PhyA. In the end, we demonstrated that only the ΔPhyA-Gal4 (ΔPhyA, 1–617 aa) variant significantly activated transgene expression. After multiple optimization strategies, the ΔPhyA-Gal4/FHY1-VP64 combination displayed high levels of induced transgene expression as well as negligible basal expression. The REDMAP system is characterized by high transcriptional activation efficiency (> 150-fold) and rapid activation/deactivation (~1 s) kinetics. Meanwhile, the transgene expression regulated by REDMAP is illumination time- and intensity-dependent. We then deployed the light sensor ΔPhyA-FHY1 (REDMAPSOS-Ras) to fine tune the Ras/Erk MAPK signaling pathway to perturb cell signaling by light. Our results imply that the REDMAP tool can be designed for diverse light-programmable tools to analyze signaling processes in cells.
Based on the REDMAP concept, we further explored a CRISPR-compatible and photoactivatable transcription system (REDMAPcas) to achieve epigenome engineering in vitro and in vivo. Our results demonstrated that the REDMAPcas system can specifically activate the transcription of user-defined endogenous genes in human cells and in mouse organs.
To explore the potential clinical application of REDMAP for gene and cell therapy, we packaged it into an AAV vector and tested its performance in mice. As expected, reporter gene expression was significantly activated upon illumination for up to 11 weeks. To validate the feasibility of REDMAP-mediated therapeutic protein release in animals based on a cell therapy scenario, HEK-293 cells engineered with REDMAP were microencapsulated and subcutaneously implanted under the dorsum of mice, rats, and rabbits. We observed significant reporter production in these animals exposed to red light. Furthermore, we also demonstrated the effectiveness of REDMAP in lowering blood glucose in diabetic mice and rats. We anticipate that this REDMAP-mediated “living cell factory” platform could be harnessed to produce various therapeutic outputs to achieve light-controlled treatment of diseases for long-term yet precisely controllable drug delivery.
In summary, based on the principle of protein heterodimerization, the REDMAP system is capable of precisely and efficiently controlling transgene expression in a remote and noninvasive way. We anticipate that the rapid activation/deactivation kinetics, high tunability, small construct size, and spatiotemporally programmable expression characteristics make the REDMAP system a valuable optogenetic tool for use in mammals to accelerate the progress of optogenetics from fundamental studies toward biomedical translational research.
1 Ye, H. & Fussenegger, M. Optogenetic Medicine: Synthetic Therapeutic Solutions Precision-Guided by Light. Cold Spring Harbor perspectives in medicine 9, doi:10.1101/cshperspect.a034371 (2019).
2 Ye, H., Baba, M. D.-E., Peng, R.-W. & Fussenegger, M. A Synthetic Optogenetic Transcription Device Enhances Blood-Glucose Homeostasis in Mice. 332, 1565-1568, doi:10.1126/science.1203535 %J Science (2011).
3 Shao, J. et al. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. 9, eaal2298, doi:10.1126/scitranslmed.aal2298 %J Science Translational Medicine (2017).
4 Shao, J. et al. Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation. Proc Natl Acad Sci U S A 115, E6722-e6730, doi:10.1073/pnas.1802448115 (2018).
5 Yu, Y. et al. Engineering a far-red light-activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors. Science advances 6, eabb1777, doi:10.1126/sciadv.abb1777 (2020).
6 Wu, J. et al. A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice. Nat Commun 11, 3708, doi:10.1038/s41467-020-17530-9 (2020).
7 Zhou, Y., Kong, D., Wang, X. et al. A small and highly sensitive red/far-red optogenetic switch for applications in mammals. Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-01036-w (2021).