Curated Optogenetic Publication Database

Abstract: The tumor microenvironment is a barrier to breast cancer therapy. Cancer-associated fibroblast cells (CAFs) can support tumor proliferation, metastasis, and drug resistance by secreting various cytokines and growth factors. Abnormal angiogenesis provides sufficient nutrients for tumor proliferation. Considering that CAFs express the sigma receptor (which recognizes anisamide, AA), we developed a CAFs and breast cancer cells dual-targeting nano drug delivery system to transport the LightOn gene express system, a spatiotemporal controlled gene expression consisting of a light-sensitive transcription factor and a specific minimal promoter. We adopted RGD (Arg-Gly-Asp) to selectively bind to the αvβ3 integrin on activated vascular endothelial cells and tumor cells. After the LightOn system has reached the tumor site, LightOn gene express system can spatiotemporal controllably express toxic Pseudomonas exotoxin An under blue light irradiation. The LightOn gene express system, combined with multifunctional nanoparticles, achieved high targeting delivery efficiency both in vitro and in vivo. It also displayed strong tumor and CAFs inhibition, anti-angiogenesis ability and anti-metastasis ability, with good safety. Moreover, it improved survival rate, survival time, and lung metastasis rate in a mouse breast cancer model. This study proves the efficacy of combining the LightOn system with targeted multifunctional nanoparticles in tumor and anti-metastatic therapy and provides new insights into tumor microenvironment regulation.

Abstract: RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR-Cas system to optogenetic control. The protocols typically take 2-3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR-Cas functions.

Abstract: Optogenetics tools for precise temporal and spatial control of protein abundance are valuable in studying diverse complex biological processes. In the present study, we engineer a monomeric tag of stabilization upon light induction (SULI) for yeast and zebrafish based on a single light-oxygen-voltage domain from Neurospora crassa. Proteins of interest fused with SULI are stable upon light illumination but are readily degraded after transfer to dark conditions. SULI shows a high dynamic range and a high tolerance to fusion at different positions of the target protein. Further studies reveal that SULI-mediated degradation occurs through a lysine ubiquitination-independent proteasome pathway. We demonstrate the usefulness of SULI in controlling the cell cycle in yeast and regulating protein stability in zebrafish, respectively. Overall, our data indicate that SULI is a simple and robust tool to quantitatively and spatiotemporally modulate protein levels for biotechnological or biomedical applications.

Abstract: The light-regulated Gal4-UAS system has offered new ways to control cellular activities with precise spatial and temporal resolution in zebrafish and Drosophila. However, the existing optogenetic Gal4-UAS systems suffer from having multiple protein components and a dependence on extraneous light-sensitive cofactors, which increase the technical complexity and limit the portability of these systems. To overcome these limitations, we herein describe the development of a novel optogenetic Gal4-UAS system (ltLightOn) for both zebrafish and Drosophila based on a single light-switchable transactivator, termed GAVPOLT, which dimerizes and binds to gene promoters to activate transgene expression upon blue light illumination. The ltLightOn system is independent of exogenous cofactors and exhibits a more than 2400-fold ON/OFF gene expression ratio, allowing quantitative, spatial, and temporal control of gene expression. We further demonstrate the usefulness of the ltLightOn system in regulating zebrafish embryonic development by controlling the expression of lefty1 by light. We believe that this single-component optogenetic system will be immensely useful in understanding the gene function and behavioral circuits in zebrafish and Drosophila.

Abstract: Individualized immunotherapy has attracted great attention due to its high specificity, effectiveness, and safety. We used an exogenous antigen to label tumor cells with MHC I molecules, which allowed neoantigen-specific T cells to recognize and kill tumor cells. A neoantigen vaccine alone cannot achieve complete tumor clearance due to a tumor immunosuppressive microenvironment. The LightOn system was developed to effectively eliminate tumor cells through the spatiotemporally controllable expression of diphtheria toxin A fragment, leading to antigen release in the tumor region. These antigens stimulated and enhanced immunological function and thus, recruited neoantigen-specific T cells to infiltrate tumor tissue. Using the nanoparticle delivery system, neoantigens produced higher delivery efficiency to lymph nodes and improved tumor targeting ability for tumor cell labelling. Good tumor inhibition and prolonged survival were achieved, while eliciting a strong immune response. The combination of a spatiotemporally controllable transgene system with tumor neoantigen labeling has great potential for tumor immunotherapy.

Abstract: Abstract not available.

Abstract: Breast cancer is a common malignancy in women. The abnormally dense collagen network in breast cancer forms a therapeutic barrier that hinders the penetration and anti-tumor effect of drugs. To overcome this hurdle, we adopted a therapeutic strategy to treat breast cancer which combined a light-switchable transgene system and losartan. The light-switchable transgene system could regulate expression of the diphtheria toxin A fragment (DTA) gene with a high on/off ratio under blue light and had great potential for spatiotemporally controllable gene expression. We developed a nanoparticle drug delivery system to achieve tumor microenvironment-responsive and targeted delivery of DTA-encoded plasmids (pDTA) to tumor sites via dual targeting to cluster of differentiation-44 and αvβ3 receptors. In vivo studies indicated that the combination of pDTA and losartan reduce the concentration of collagen type I from 5.9 to 1.9 µg/g and decreased the level of active transforming growth factor-β by 75.0% in tumor tissues. Moreover, deeper tumor penetration was achieved, tumor growth was inhibited, and the survival rate was increased. Our combination strategy provides a novel and practical method for clinical treatment of breast cancer.

Abstract: RNA-binding proteins (RBPs) play an essential role in regulating the function of RNAs in a cellular context, but our ability to control RBP activity in time and space is limited. Here, we describe the engineering of LicV, a photoswitchable RBP that binds to a specific RNA sequence in response to blue light irradiation. When fused to various RNA effectors, LicV allows for optogenetic control of RNA localization, splicing, translation and stability in cell culture. Furthermore, LicV-assisted CRISPR-Cas systems allow for efficient and tunable photoswitchable regulation of transcription and genomic locus labeling. These data demonstrate that the photoswitchable RBP LicV can serve as a programmable scaffold for the spatiotemporal control of synthetic RNA effectors.

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: A light-switchable transgene system called LightOn gene expression system could regulate gene expression with a high on/off ratio under blue light, and have great potential for spatiotemporally controllable gene expression. We developed a nanoparticle drug delivery system (NDDS) to achieve tumor microenvironment-responsive and targeted delivery of diphtheria toxin A (DTA) fragment-encoded plasmids to tumor sites. The expression of DTA was induced by exposure to blue light. Nanoparticles composed of polyethylenimine and vitamin E succinate linked by a disulfide bond, and PEGylated hyaluronic acid modified with RGD peptide, accumulated in tumor tissues and were actively internalized into 4T1 cells via dual targeting to CD44 and αvβ3 receptors. The LightOn gene expression system was able to control target protein expression through regulation of the intensity or duration of blue light exposure. In vitro studies showed that light-induced DTA expression reduced 4T1 cell viability and induced apoptosis. Furthermore, the LightOn gene expression system enabled spatiotemporal control of the expression of DTA in a mouse 4T1 tumor xenograft model, which resulted in excellent antitumor effects, reduced tumor angiogenesis, and no systemic toxicity. The combination of the LightOn gene expression system and NDDS may be an effective strategy for treatment of breast cancer.

Abstract: Light-regulated modules offer unprecedented new ways to control cellular behaviour with precise spatial and temporal resolution. Among a variety of bacterial light-switchable gene expression systems, single-component systems consisting of single transcription factors would be more useful due to the advantages of speed, simplicity, and versatility. In the present study, we developed a single-component light-activated bacterial gene expression system (eLightOn) based on a novel LOV domain from Rhodobacter sphaeroides (RsLOV). The eLightOn system showed significant improvements over the existing single-component bacterial light-activated expression systems, with benefits including a high ON/OFF ratio of >500-fold, a high activation level, fast activation kinetics, and/or good adaptability. Additionally, the induction characteristics, including regulatory windows, activation kinetics and light sensitivities, were highly tunable by altering the expression level of LexRO. We demonstrated the usefulness of the eLightOn system in regulating cell division and swimming by controlling the expression of the FtsZ and CheZ genes, respectively, as well as constructing synthetic Boolean logic gates using light and arabinose as the two inputs. Taken together, our data indicate that the eLightOn system is a robust and highly tunable tool for quantitative and spatiotemporal control of bacterial gene expression.

Abstract: Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Here, we develop Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA's transcription, localization and translation. Quantification of the levels of proteins and their messenger RNAs in single cells suggests that translation is governed by normal enzyme kinetics but with marked heterogeneity. We further show that Peppers can be used for imaging genomic loci with CRISPR display, for real-time tracking of protein-RNA tethering, and for super-resolution imaging. We believe these FRs will be useful tools for live imaging of cellular RNAs.

Abstract: Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In this study, we developed a single-component light-switchable gene expression system for yeast cells, termed yLightOn system. The yLightOn system is independent of exogenous cofactors, and exhibits more than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics, and high spatial resolution. We demonstrated the usefulness of the yLightOn system in regulating cell growth and cell cycle by stringently controlling the expression of His3 and ΔN Sic1 genes, respectively. Furthermore, we engineered a bidirectional expression module that allows the simultaneous control of the expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative, and spatially specific degradation of ssrA-tagged protein was observed. We suggest that this single-component optogenetic system will be immensely helpful in understanding cellular gene regulatory networks and in the design of robust genetic circuits for synthetic biology.

Abstract: Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies.

Abstract: Light-switchable gene expression systems provide transient, non-invasive and reversible means to control biological processes with high tunability and spatiotemporal resolution. In bacterial cells, a few light-regulated gene expression systems based on photoreceptors and two-component regulatory systems (TCSs) have been reported, which respond to blue, green or red light.

Abstract: Programmable transcription factors can enable precise control of gene expression triggered by a chemical inducer or light. To obtain versatile transgene system with combined benefits of a chemical inducer and light inducer, we created various chimeric promoters through the assembly of different copies of the tet operator and Gal4 operator module, which simultaneously responded to a tetracycline-responsive transcription factor and a light-switchable transactivator. The activities of these chimeric promoters can be regulated by tetracycline and blue light synergistically or antagonistically. Further studies of the antagonistic genetic circuit exhibited high spatiotemporal resolution and extremely low leaky expression, which therefore could be used to spatially and stringently control the expression of highly toxic protein Diphtheria toxin A for light regulated gene therapy. When transferring plasmids engineered for the gene switch-driven expression of a firefly luciferase (Fluc) into mice, the Fluc expression levels of the treated animals directly correlated with the tetracycline and light input program. We suggest that dual-input genetic circuits using TET and light that serve as triggers to achieve expression profiles may enable the design of robust therapeutic gene circuits for gene- and cell-based therapies.

Abstract: Several light-regulated genetic circuits have been applied to spatiotemporally control transgene expression in mammalian cells. However, simultaneous regulation of multiple genes using one genetic device by light has not yet been reported. In this study, we engineered a bidirectional expression module based on LightOn system. Our data showed that both reporter genes could be regulated at defined and quantitative levels. Simultaneous regulation of four genes was further achieved in cultured cells and mice. Additionally, we successfully utilized the bidirectional expression module to monitor the expression of a suicide gene, showing potential for photodynamic gene therapy. Collectively, we provide a robust and useful tool to simultaneously control multiple genes expression by light, which will be widely used in biomedical research and biotechnology.

Abstract: Spatiotemporal control of transgene expression in living cells provides new opportunities for the characterization of gene function in complex biological processes. We previously reported a synthetic, light-switchable transgene expression system called LightOn that can be used to control gene expression using blue light. In the present study, we modified the different promoter segments of the light switchable transcription factor GAVPO and the target gene, and assayed their effects on protein expression under dark or light conditions. The results showed that the LightOn system maintained its high on/off ratio under most modifications, but its induction efficiency and background gene expression level can be fine-tuned by modifying the core promoter, the UASG sequence number, the length of the spacer between UASG and the core promoter of the target protein, and the expression level of the GAVPO transcription factor. Thus, the LightOn gene expression system can be adapted to a large range of applications according to the requirements of the background and the induced gene expression.

Abstract: We developed a light-switchable transgene system based on a synthetic, genetically encoded light-switchable transactivator. The transactivator binds promoters upon blue-light exposure and rapidly initiates transcription of target transgenes in mammalian cells and in mice. This transgene system provides a robust and convenient way to spatiotemporally control gene expression and can be used to manipulate many biological processes in living systems with minimal perturbation.