Curated Optogenetic Publication Database

Abstract: Red light optogenetic systems are in high demand for the precise control of gene expression for gene- and cell-based therapies. Here, we report a red/far-red light-inducible photoswitch (REDLIP) system based on the chimeric photosensory protein FnBphP (Fn-REDLIP) or PnBphP (Pn-REDLIP) and their interaction partner LDB3, which enables efficient dynamic regulation of gene expression with a timescale of seconds without exogenous administration of a chromophore in mammals. We used the REDLIP system to establish the REDLIP-mediated CRISPR-dCas9 (REDLIPcas) system, enabling optogenetic activation of endogenous target genes in mammalian cells and mice. The REDLIP system is small enough to support packaging into adeno-associated viruses (AAVs), facilitating its therapeutic application. Demonstrating its capacity to treat metabolic diseases, we show that an AAV-delivered Fn-REDLIP system achieved optogenetic control of insulin expression to effectively lower blood glucose levels in type 1 diabetes model mice and control an anti-obesity therapeutic protein (thymic stromal lymphopoietin, TSLP) to reduce body weight in obesity model mice. REDLIP is a compact and sensitive optogenetic tool for reversible and non-invasive control that can facilitate basic biological and biomedical research.

Abstract: Synthetic biology applications require finely tuned gene expression, often mediated by synthetic transcription factors (sTFs) compatible with the human genome and transcriptional regulation mechanisms. While various DNA-binding and activation domains have been developed for different applications, advanced artificially controllable sTFs with improved regulatory capabilities are required for increasingly sophisticated applications. Here, in mammalian cells and mice, we validate the transactivator function and homo-/heterodimerization activity of the plant-derived phytochrome chaperone proteins, FHY1 and FHL. Our results demonstrate that FHY1/FHL form a photosensing transcriptional regulation complex (PTRC) through interaction with the phytochrome, ΔPhyA, that can toggle between active and inactive states through exposure to red or far-red light, respectively. Exploiting this capability, we develop a light-switchable platform that allows for orthogonal, modular, and tunable control of gene transcription, and incorporate it into a PTRC-controlled CRISPRa system (PTRCdcas) to modulate endogenous gene expression. We then integrate the PTRC with small molecule- or blue light-inducible regulatory modules to construct a variety of highly tunable systems that allow rapid and reversible control of transcriptional regulation in vitro and in vivo. Validation and deployment of these plant-derived phytochrome chaperone proteins in a PTRC platform have produced a versatile, powerful tool for advanced research and biomedical engineering applications.

Abstract: Diabetes mellitus and its associated secondary complications have become a pressing global healthcare issue. The current integrated theranostic plan involves a glucometer-tandem pump. However, external condition-responsive insulin delivery systems utilizing rigid glucose sensors pose challenges in on-demand, long-term insulin administration. To overcome these challenges, we present a novel model of antidiabetic management based on printable metallo-nucleotide hydrogels and optogenetic engineering. The conductive hydrogels were self-assembled by bioorthogonal chemistry using oligonucleotides, carbon nanotubes, and glucose oxidase, enabling continuous glucose monitoring in a broad range (0.5-40 mM). The optogenetically engineered cells were enabled glucose regulation in type I diabetic mice via a far-red light-induced transgenic expression of insulin with a month-long avidity. Combining with a microchip-integrated microneedle patch, a prototyped close-loop system was constructed. The glucose levels detected by the sensor were received and converted by a wireless controller to modulate far-infrared light, thereby achieving on-demand insulin expression for several weeks. This study sheds new light on developing next-generation diagnostic and therapy systems for personalized and digitalized precision medicine.

Abstract: Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

Abstract: Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.

Abstract: Secreted reporters have been demonstrated to be simple and useful tools for analyzing transcriptional regulation in mammalian cells. The distinctive feature of these assays is the ability to detect reporter gene expression in the culture supernatant without affecting the cell physiology or leading to cell lysis, which allows repeated experimentation and sampling of the culture medium using the same cell cultures. Secreted embryonic alkaline phosphatase (SEAP) is one of the most widely used reporter, which can be easily detected using colorimetry following incubation with a substrate, such as p-nitrophenol phosphate. In this report, we present detailed procedures for detection and quantification of the SEAP reporter. We believe that this step-by-step protocol can be easily used by researchers to monitor and measure molecular genetic events in a variety of mammalian cells due to its simplicity and ease of handling. Graphical abstract Schematic overview of the workflow described in this protocol.

Abstract: Pluripotent stem cells (PSCs) hold great promise for cell-based therapies, disease modeling, and drug discovery. Classic somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) is often achieved based on overexpression of transcription factors (TFs). However, this process is limited by side effect of overexpressed TFs and unpredicted targeting of TFs. Pinpoint control over endogenous TFs expression can provide the ability to reprogram cell fate and tissue function. Here, a light-inducible cell reprogramming (LIRE) system is developed based on a photoreceptor protein cryptochrome system and clustered regularly interspaced short palindromic repeats/nuclease-deficient CRISPR-associated protein 9 for induced PSCs reprogramming. This system enables remote, non-invasive optogenetical regulation of endogenous Sox2 and Oct4 loci to reprogram mouse embryonic fibroblasts into iPSCs (iPSCLIRE ) under light-emitting diode-based illumination. iPSCLIRE cells can be efficiently differentiated into different cells by upregulating a corresponding TF. iPSCLIRE cells are used for blastocyst injection and optogenetic chimeric mice are successfully generated, which enables non-invasive control of user-defined endogenous genes in vivo, providing a valuable tool for facile and traceless controlled gene expression studies and genetic screens in mice. This LIRE system offers a remote, traceless, and non-invasive approach for cellular reprogramming and modeling of complex human diseases in basic biological research and regenerative medicine applications.

Abstract: Surgical resection is the main treatment option for most solid tumors, yet cancer recurrence after surgical resection remains a significant challenge in cancer therapy. Recent advances in cancer immunotherapy are enabling radical cures for many tumor patients, but these technologies remain challenging to apply because of side effects related to uncontrollable immune system activation. Here, we develop far-red light-controlled immunomodulatory engineered cells (FLICs) that we load into a hydrogel scaffold, enabling the precise optogenetic control of cytokines release (IFN-β, TNF-α, and IL-12) upon illumination. Experiments with a B16F10 melanoma resection mouse model show that FLICs-loaded hydrogel implants placed at the surgical wound site achieve sustainable release of immunomodulatory cytokines, leading to prevention of tumor recurrence and increased animal survival. Moreover, the FLICs-loaded hydrogel implants elicit long-term immunological memory that prevents against tumor recurrence. Our findings illustrate that this optogenetic perioperative immunotherapy with FLICs-loaded hydrogel implants offers a safe treatment option for solid tumors based on activating host innate and adaptive immune systems to inhibit tumor recurrence after surgery. Beyond extending the optogenetics toolbox for immunotherapy, we envision that our optogenetic-controlled living cell factory platform could be deployed for other biomedical contexts requiring precision induction of bio-therapeutic dosage.

Abstract: Gene- and cell-based therapies are the next frontiers in the field of medicine. Both are transformative and innovative therapies; however, a lack of safety data limits the translation of such promising technologies to the clinic. Improving the safety and promoting the clinical translation of these therapies can be achieved by tightly regulating the release and delivery of therapeutic outputs. In recent years, the rapid development of optogenetic technology has provided opportunities to develop precision-controlled gene- and cell-based therapies, in which light is introduced to precisely and spatiotemporally manipulate the behaviour of genes and cells. This review focuses on the development of optogenetic tools and their applications in biomedicine, including photoactivated genome engineering and phototherapy for diabetes and tumours. The prospects and challenges of optogenetic tools for future clinical applications are also discussed.

Abstract: Diabetes treatment and rehabilitation are usually a lifetime process. Optogenetic engineered designer cell-therapy holds great promise in regulating blood glucose homeostasis. However, portable, sustainable, and long-term energy supplementation has previously presented a challenge for the use of optogenetic stimulation in vivo. Herein, we purpose a self-powered optogenetic system (SOS) for implantable blood glucose control. The SOS consists of a biocompatible far-red light (FRL) source, FRL-triggered transgene-expressing cells, a power management unit, and a flexible implantable piezoelectric nanogenerator (i-PENG) to supply long-term energy by converting biomechanical energy into electricity. Our results show that this system can harvest energy from body movement and power the FRL source, which then significantly enhanced production of a short variant of human glucagon-like peptide 1 (shGLP-1) in vitro and in vivo. Indeed, diabetic mice equipped with the SOS showed rapid restoration of blood glucose homeostasis, improved glucose, and insulin tolerance. Our results suggest that the SOS is sufficiently effective in self-powering the modulation of therapeutic outputs to control glucose homeostasis and, furthermore, present a new strategy for providing energy in optogenetic-based cell therapy.

Abstract: The CRISPR-Cas12a has been harnessed as a powerful tool for manipulating targeted gene expression. The possibility to manipulate the activity of CRISPR-Cas12a with a more precise spatiotemporal resolution and deep tissue permeability will enable targeted genome engineering and deepen our understanding of the gene functions underlying complex cellular behaviors. However, currently available inducible CRISPR-Cas12a systems are limited by diffusion, cytotoxicity, and poor tissue permeability. Here, we developed a far-red light (FRL)–inducible CRISPR-Cas12a (FICA) system that can robustly induce gene editing in mammalian cells, and an FRL-inducible CRISPR-dCas12a (FIdCA) system based on the protein-tagging system SUperNova (SunTag) that can be used for gene activation under light-emitting diode–based FRL. Moreover, we show that the FIdCA system can be deployed to activate target genes in mouse livers. These results demonstrate that these systems developed here provide robust and efficient platforms for programmable genome manipulation in a noninvasive and spatiotemporal fashion.

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.

Abstract: Diabetes affects almost half a billion people, and all individuals with type 1 diabetes (T1D) and a large portion of individuals with type 2 diabetes rely on self-administration of the peptide hormone insulin to achieve glucose control. However, this treatment modality has cumbersome storage and equipment requirements and is susceptible to fatal user error. Here, reasoning that a cell-based therapy could be coupled to an external induction circuit for blood glucose control, as a proof of concept we developed far-red light (FRL)-activated human islet-like designer (FAID) cells and demonstrated how FAID cell implants achieved safe and sustained glucose control in diabetic model mice. Specifically, by introducing a FRL-triggered optogenetic device into human mesenchymal stem cells (hMSCs), which we encapsulated in poly-(l-lysine)-alginate and implanted subcutaneously under the dorsum of T1D model mice, we achieved FRL illumination-inducible secretion of insulin that yielded improvements in glucose tolerance and sustained blood glucose control over traditional insulin glargine treatment. Moreover, the FAID cell implants attenuated both oxidative stress and development of multiple diabetes-related complications in kidneys. This optogenetics-controlled "living cell factory" platform could be harnessed to develop multiple synthetic designer therapeutic cells to achieve long-term yet precisely controllable drug delivery.

Abstract: Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

Abstract: Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.

Abstract: With the development of mobile communication technology, smartphones have been used in point-of-care technologies (POCTs) as an important part of telemedicine. Using a multidisciplinary design principle coupling electrical engineering, software development, synthetic biology, and optogenetics, the investigators developed a smartphone-controlled semiautomatic theranostic system that regulates blood glucose homeostasis in diabetic mice in an ultraremote-control manner. The present chapter describes how the investigators tailor-designed the implant architecture "HydrogeLED," which is capable of coharboring a designer-cell-carrying alginate hydrogel and wirelessly powered far-red light LEDs. Using diabetes mellitus as a model disease, the in vivo expression of insulin or human glucagon-like peptide 1 (shGLP-1) from HydrogeLED implants could be controlled not only by pre-set ECNU-TeleMed programs, but also by a custom-engineered Bluetooth-active glucometer in a semiautomatic and glycemia-dependent manner. As a result, blood glucose homeostasis was semiautomatically maintained in diabetic mice through the smartphone-controlled semiautomatic theranostic system. By combining digital signals with optogenetically engineered cells, the present study provides a new method for the integrated diagnosis and treatment of diseases.

Abstract: With the increasing indispensable role of smartphones in our daily lives, the mobile health care system coupled with embedded physical sensors and modern communication technologies make it an attractive technology for enabling the remote monitoring of an individual's health. Using a multidisciplinary design principle coupled with smart electronics, software, and optogenetics, the investigators constructed smartphone-controlled optogenetic switches to enable the ultraremote-control transgene expression. A custom-designed SmartController system was programmed to process wireless signals from smartphones, enabling the regulation of therapeutic outputs production by optically engineered cells via a far-red light (FRL)-responsive optogenetic interface. In the present study, the investigators describe the details of the protocols for constructing smartphone-controlled optogenetic switches, including the rational design of an FRL-triggered transgene expression circuit, the procedure for cell culture and transfection, the implementation of the smartphone-controlled far-red light-emitting diode (LED) module, and the reporter detection assay.

Abstract: Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

Abstract: Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

Abstract: The Cre-loxP recombination system is a powerful tool for genetic manipulation. However, there are widely recognized limitations with chemically inducible Cre-loxP systems, and the UV and blue-light induced systems have phototoxicity and minimal capacity for deep tissue penetration. Here, we develop a far-red light-induced split Cre-loxP system (FISC system) based on a bacteriophytochrome optogenetic system and split-Cre recombinase, enabling optogenetical regulation of genome engineering in vivo solely by utilizing a far-red light (FRL). The FISC system exhibits low background and no detectable photocytotoxicity, while offering efficient FRL-induced DNA recombination. Our in vivo studies showcase the strong organ-penetration capacity of FISC system, markedly outperforming two blue-light-based Cre systems for recombination induction in the liver. Demonstrating its strong clinical relevance, we successfully deploy a FISC system using adeno-associated virus (AAV) delivery. Thus, the FISC system expands the optogenetic toolbox for DNA recombination to achieve spatiotemporally controlled, non-invasive genome engineering in living systems.

Abstract: It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)–activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode–based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.

Abstract: Gene- and cell-based therapies are well recognized as central pillars of next-generation medicine, but controllability remains a critical issue for clinical applications. In this context, optogenetics is opening up exciting new opportunities for precision-guided medicine by using illumination with light of appropriate intensity and wavelength as a trigger signal to achieve pinpoint spatiotemporal control of cellular activities, such as transgene expression. In this review, we highlight recent advances in optogenetics, focusing on devices for biomedical applications. We introduce the construction and applications of optogenetic-based biomedical tools to treat neurological diseases, diabetes, heart diseases, and cancer, as well as bioelectronic implants that combine light-interfaced electronic devices and optogenetic systems into portable personalized precision bioelectronic medical tools. Optogenetics-based technology promises the capability to achieve traceless, remotely controlled precision dosing of an enormous range of therapeutic outputs. Finally, we discuss the prospects for optogenetic medicine, as well as some emerging challenges.

Abstract: The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.

Abstract: With the increasingly dominant role of smartphones in our lives, mobile health care systems integrating advanced point-of-care technologies to manage chronic diseases are gaining attention. Using a multidisciplinary design principle coupling electrical engineering, software development, and synthetic biology, we have engineered a technological infrastructure enabling the smartphone-assisted semiautomatic treatment of diabetes in mice. A custom-designed home server SmartController was programmed to process wireless signals, enabling a smartphone to regulate hormone production by optically engineered cells implanted in diabetic mice via a far-red light (FRL)-responsive optogenetic interface. To develop this wireless controller network, we designed and implanted hydrogel capsules carrying both engineered cells and wirelessly powered FRL LEDs (light-emitting diodes). In vivo production of a short variant of human glucagon-like peptide 1 (shGLP-1) or mouse insulin by the engineered cells in the hydrogel could be remotely controlled by smartphone programs or a custom-engineered Bluetooth-active glucometer in a semiautomatic, glucose-dependent manner. By combining electronic device-generated digital signals with optogenetically engineered cells, this study provides a step toward translating cell-based therapies into the clinic.