Showing 1 - 25 of 81 results
Light induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.
There is currently a lack of tools capable of perturbing genes in both a precise and spatiotemporal fashion. CRISPR’s ease of use and flexibility, coupled with light’s unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here we present a new optogenetic CRISPR tool, BLU-VIPR, that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of gRNA production. This simplifies spatiotemporal gene perturbation and works in vivo with cells previously intractable to optogenetic gene editing. We engineered BLU-VIPR around a new potent blue-light activated transcription factor and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single mRNA transcript, allowing for optogenetic gene editing in T lymphocytes in vivo.
Photoactivatable base editors for spatiotemporally controlled genome editing in vivo.
CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.
Optogenetic engineering of STING signaling allows remote immunomodulation to enhance cancer immunotherapy.
The cGAS-STING signaling pathway has emerged as a promising target for immunotherapy development. Here, we introduce a light-sensitive optogenetic device for control of the cGAS/STING signaling to conditionally modulate innate immunity, called 'light-inducible SMOC-like repeats' (LiSmore). We demonstrate that photo-activated LiSmore boosts dendritic cell (DC) maturation and antigen presentation with high spatiotemporal precision. This non-invasive approach photo-sensitizes cytotoxic T lymphocytes to engage tumor antigens, leading to a sustained antitumor immune response. When combined with an immune checkpoint blocker (ICB), LiSmore improves antitumor efficacy in an immunosuppressive lung cancer model that is otherwise unresponsive to conventional ICB treatment. Additionally, LiSmore exhibits an abscopal effect by effectively suppressing tumor growth in a distal site in a bilateral mouse model of melanoma. Collectively, our findings establish the potential of targeted optogenetic activation of the STING signaling pathway for remote immunomodulation in mice.
Opto4E-BP, an optogenetic tool for inducible, reversible, and cell type-specific inhibition of translation initiation.
The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is one of the primary triggers for initiating cap-dependent translation. Amongst its functions, mTORC1 phosphorylates eIF4E-binding proteins (4E-BPs), which prevents them from binding to eIF4E and thereby enables translation initiation. mTORC1 signaling is required for multiple forms of protein synthesis- dependent synaptic plasticity and various forms of long-term memory (LTM), including associative threat memory. However, the approaches used thus far to target mTORC1 and its effectors, such as pharmacological inhibitors or genetic knockouts, lack fine spatial and temporal control. The development of a conditional and inducible eIF4E knockdown mouse line partially solved the issue of spatial control, but still lacked optimal temporal control to study memory consolidation. Here, we have designed a novel optogenetic tool (Opto4E-BP) for cell type-specific, light-dependent regulation of eIF4E in the brain. We show that light-activation of Opto4E-BP decreases protein synthesis in HEK cells and primary mouse neurons. In situ, light-activation of Opto4E-BP in excitatory neurons decreased protein synthesis in acute amygdala slices. Finally, light activation of Opto4E-BP in principal excitatory neurons in the lateral amygdala (LA) of mice after training blocked the consolidation of LTM. The development of this novel optogenetic tool to modulate eIF4E-dependent translation with spatiotemporal precision will permit future studies to unravel the complex relationship between protein synthesis and the consolidation of LTM.
RNA G-quadruplexes forming scaffolds for alpha-synuclein aggregation lead to progressive neurodegeneration.
Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are primarily neurodegenerative diseases with progressive decline in motor function. Aggregates composed of alpha-synuclein, which are known as Lewy bodies, are a neuropathological hallmark of synucleinopathies; their pathogenesis has been attributed to neuronal loss owing to intracellular alpha-synuclein accumulation. However, the mechanism of alpha-synuclein aggregation remains unclear. Here we show that the RNA G-quadruplexes assembly forms scaffolds for alpha-synuclein aggregation, thereby contributing to neurodegeneration. RNA G-quadruplexes undergo phase separation and form scaffolds for co-aggregation with & alpha-synuclein. Upon pathogenic alpha-synuclein seeds-induced cellular stress and an optogenetic assembly of RNA G-quadruplexes, phase-separated RNA G-quadruplexes served as scaffolds for & alpha-synuclein phase transition, and the co-aggregates initiated synaptic dysfunction and Parkinsonism in mice. Treatment with 5-aminolevulinic acid and protoporphyrin IX, which prevents RNA G-quadruplexes phase separation, attenuates alpha-synuclein phase transition, neurodegeneration, and motor deficits in synucleinopathy model mice. Together, the RNA G-quadruplexes assembly accelerates alpha-synuclein phase transition and aggregation owing to intracellular Ca2+ homeostasis, thereby contributing to the pathogenesis of synucleinopathies.
A Bioluminescent Activity Dependent (BLADe) Platform for Converting Neuronal Activity to Photoreceptor Activation.
We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca2+) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains. Critical features of the converter luciferase are light emission low enough to not activate photoreceptors under baseline condition and high enough to activate photosensing elements in the presence of Ca2+ and luciferin. We demonstrate performance of this activity-dependent sensor and integrator for changing membrane potential and driving transcription in individual and populations of neurons in vitro and in vivo.
Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.
The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.
Light-stimulated insulin secretion from pancreatic islet-like organoids derived from human pluripotent stem cells.
Optogenetic techniques permit non-invasive, spatiotemporal, and reversible modulation of cellular activities. Here, we report a novel optogenetic regulatory system for insulin secretion in human pluripotent stem cell (hPSC)-derived pancreatic islet-like organoids using monSTIM1 (monster-opto-Stromal interaction molecule 1), an ultra-light-sensitive OptoSTIM1 variant. The monSTIM1 transgene was incorporated at the AAVS1 locus in human embryonic stem cells (hESCs) by CRISPR-Cas9-mediated genome editing. Not only were we able to elicit light-induced intracellular Ca2+ concentration ([Ca2+]i) transients from the resulting homozygous monSTIM1+/+-hESCs, but we also successfully differentiated them into pancreatic islet-like organoids (PIOs). Upon light stimulation, the β-cells in these monSTIM1+/+-PIOs displayed reversible and reproducible [Ca2+]i transient dynamics. Furthermore, in response to photoexcitation, they secreted human insulin. Light-responsive insulin secretion was similarly observed in monSTIM1+/+-PIOs produced from neonatal diabetes (ND) patient-derived induced pluripotent stem cells (iPSCs). Under LED illumination, monSTIM1+/+-PIO-transplanted diabetic mice produced human c-peptide. Collectively, we developed a cellular model for the optogenetic control of insulin secretion using hPSCs, with the potential to be applied to the amelioration of hyperglycemic disorders.
Spatiotemporally controllable diphtherin transgene system and neoantigen immunotherapy.
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.
Near-infrared-inducible Bcl-2-associated X protein system for apoptosis regulation in vivo.
Bcl-2-associated X protein (BAX) plays a vital role in maintaining tissue homeostasis and participates in the pathogenesis of various diseases. Poor spatiotemporal control remains a challenge in direct pharmacological modulation and genetic perturbation of BAX’s activity. Herein, we developed a near-infrared (NIR) light-inducible BAX (NiBAX) system that enabled remote and spatiotemporal control of BAX-mediated apoptosis. The NiBAX was constructed by integration of two independent modules: blue light-responsive optogenetics BAX plasmids for regulating migration of BAX to mitochondria and upconversion nanoparticles-encapsulated flexible implant for converting tissue-penetrative NIR light into blue light. This NiBAX could readily induce robust BAX-based cellular apoptosis in vitro, and elicit effective apoptosis-mediated oncotherapy in vivo under NIR light. Collectively, the upconversion optogenetic NiBAX system provides an advanced tool for BAX-related cellular behavior control.
Enhancement of Vivid-based Photo-Activatable Gal4 Transcription Factor in Mammalian Cells.
The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.
A doxycycline- and light-inducible Cre recombinase mouse model for optogenetic genome editing.
The experimental need to engineer the genome both in time and space, has led to the development of several photoactivatable Cre recombinase systems. However, the combination of inefficient and non-intentional background recombination has prevented thus far the wide application of these systems in biological and biomedical research. Here, we engineer an optimized photoactivatable Cre recombinase system that we refer to as doxycycline- and light-inducible Cre recombinase (DiLiCre). Following extensive characterization in cancer cell and organoid systems, we generate a DiLiCre mouse line, and illustrated the biological applicability of DiLiCre for light-induced mutagenesis in vivo and positional cell-tracing by intravital microscopy. These experiments illustrate how newly formed HrasV12 mutant cells follow an unnatural movement towards the interfollicular dermis. Together, we develop an efficient photoactivatable Cre recombinase mouse model and illustrate how this model is a powerful genome-editing tool for biological and biomedical research.
Stable Transgenic Mouse Strain with Enhanced Photoactivatable Cre Recombinase for Spatiotemporal Genome Manipulation.
Optogenetic genome engineering is a powerful technology for high-resolution spatiotemporal genetic manipulation, especially for in vivo studies. It is difficult to generate stable transgenic animals carrying a tightly regulated optogenetic system, as its long-term expression induces high background activity. Here, the generation of an enhanced photoactivatable Cre recombinase (ePA-Cre) transgenic mouse strain with stringent light responsiveness and high recombination efficiency is reported. Through serial optimization, ePA-Cre is developed to generate a transgenic mouse line that exhibits 175-fold induction upon illumination. Efficient light-dependent recombination is detected in embryos and various adult tissues of ePA-Cre mice crossed with the Ai14 tdTomato reporter. Importantly, no significant background Cre activity is detected in the tested tissues except the skin. Moreover, efficient light-inducible cell ablation is achieved in ePA-Cre mice crossed with Rosa26-LSL-DTA mice. In conclusion, ePA-Cre mice offer a tightly inducible, highly efficient, and spatiotemporal-specific genome engineering tool for multiple applications.
A general approach for engineering RTKs optically controlled with far-red light.
Regulation of receptor tyrosine kinase (RTK) activity is necessary for studying cell signaling pathways in health and disease. We developed a generalized approach for engineering RTKs optically controlled with far-red light. We targeted the bacterial phytochrome DrBphP to the cell surface and allowed its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. Systematic optimization of these constructs has resulted in optically regulated epidermal growth factor receptor, HER2, TrkA, TrkB, FGFR1, IR1, cKIT and cMet, named eDrRTKs. eDrRTKs induced downstream signaling in mammalian cells in tens of seconds. The ability to activate eDrRTKs with far-red light enabled spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays. We validated eDrTrkB performance in mice and found that minimally invasive stimulation in the neocortex with penetrating via skull far-red light-induced neural activity, early immediate gene expression and affected sleep patterns.
Light-switchable diphtherin transgene system combined with losartan for triple negtative breast cancer therapy based on nano drug delivery system.
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.
Repetitive short-pulsed illumination efficiently activates photoactivatable-Cre as continuous illumination in embryonic stem cells and pre-implantation embryos of transgenic mouse.
The Cre-loxP system has been widely used for specific DNA recombination which induces gene inactivation or expression. Recently, photoactivatable-Cre (PA-Cre) proteins have been developed as a tool for spatiotemporal control of the enzymatic activity of Cre recombinase. Here, we generated transgenic mice bearing a PA-Cre gene and systematically investigated the conditions of photoactivation for the PA-Cre in embryonic stem cells (ESCs) derived from the transgenic mice and in a simple mathematical model. Cre-mediated DNA recombination was induced in 16% of the PA-Cre ESCs by 6 hr continuous illumination. We show that repetitive pulsed illumination efficiently induced DNA recombination with low light energy as efficient as continuous illumination in the ESCs (96 ± 15% of continuous illumination when pulse cycle was 2 s), which was also supported by a minimal mathematical model. DNA recombination by the PA-Cre was also successfully induced in the transgenic mouse pre-implantation embryos under the developed conditions. These results suggest that strategies based on repetitive pulsed illumination are efficient for the activation of photoactivatable Cre and, possibly other photo-switchable proteins.
A small and highly sensitive red/far-red optogenetic switch for applications in mammals.
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.
Single-component near-infrared optogenetic systems for gene transcription regulation.
Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. However, available NIR optogenetic systems consist of several protein components of large size and multidomain structure. Here, we engineer single-component NIR systems consisting of evolved photosensory core module of Idiomarina sp. bacterial phytochrome, named iLight, which are smaller and packable in adeno-associated virus. We characterize iLight in vitro and in gene transcription repression in bacterial and gene transcription activation in mammalian cells. Bacterial iLight system shows 115-fold repression of protein production. Comparing to multi-component NIR systems, mammalian iLight system exhibits higher activation of 65-fold in cells and faster 6-fold activation in deep tissues of mice. Neurons transduced with viral-encoded iLight system exhibit 50-fold induction of fluorescent reporter. NIR light-induced neuronal expression of green-light-activatable CheRiff channelrhodopsin causes 20-fold increase of photocurrent and demonstrates efficient spectral multiplexing.
Temporal induction of Lhx8 by optogenetic control system for efficient bone regeneration.
The spatiotemporal regulation of essential genes is crucial for controlling the growth and differentiation of cells in a precise manner during regeneration. Recently, optogenetics was considered as a potent technology for sophisticated regulation of target genes, which might be a promising tool for regenerative medicine. In this study, we used an optogenetic control system to precisely regulate the expression of Lhx8 to promote efficient bone regeneration.
Exosome-based delivery of super-repressor IκBα ameliorates kidney ischemia-reperfusion injury.
Ischemia-reperfusion injury is a major cause of acute kidney injury. Recent studies on the pathophysiology of ischemia-reperfusion-induced acute kidney injury showed that immunologic responses significantly affect kidney ischemia-reperfusion injury and repair. Nuclear factor (NF)-ĸB signaling, which controls cytokine production and cell survival, is significantly involved in ischemia-reperfusion-induced acute kidney injury, and its inhibition can ameliorate ischemic acute kidney injury. Using EXPLOR, a novel, optogenetically engineered exosome technology, we successfully delivered the exosomal super-repressor inhibitor of NF-ĸB (Exo-srIĸB) into B6 wild type mice before/after kidney ischemia-reperfusion surgery, and compared outcomes with those of a control exosome (Exo-Naïve)-injected group. Exo-srIĸB treatment resulted in lower levels of serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin in post-ischemic mice than in the Exo-Naïve treatment group. Systemic delivery of Exo-srIĸB decreased NF-ĸB activity in post-ischemic kidneys and reduced apoptosis. Post-ischemic kidneys showed decreased gene expression of pro-inflammatory cytokines and adhesion molecules with Exo-srIĸB treatment as compared with the control. Intravital imaging confirmed the uptake of exosomes in neutrophils and macrophages. Exo-srIĸB treatment also significantly affected post-ischemic kidney immune cell populations, lowering neutrophil, monocyte/macrophage, and T cell frequencies than those in the control. Thus, modulation of NF-ĸB signaling through exosomal delivery can be used as a novel therapeutic method for ischemia-reperfusion-induced acute kidney injury.
Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering.
Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.
A CRISPR-Cas9-Based Near-Infrared Upconversion-Activated DNA Methylation Editing System.
DNA methylation is a kind of a crucial epigenetic marker orchestrating gene expression, molecular function, and cellular phenotype. However, manipulating the methylation status of specific genes remains challenging. Here, a clustered regularly interspaced palindromic repeats-Cas9-based near-infrared upconversion-activated DNA methylation editing system (CNAMS) was designed for the optogenetic editing of DNA methylation. The fusion proteins of photosensitive CRY2PHR, the catalytic domain of DNMT3A or TET1, and the fusion proteins for CIBN and catalytically inactive Cas9 (dCas9) were engineered. The CNAMS could control DNA methylation editing in response to blue light, thus allowing methylation editing in a spatiotemporal manner. Furthermore, after combination with upconversion nanoparticles, the spectral sensitivity of DNA methylation editing was extended from the blue light to near-infrared (NIR) light, providing the possibility for remote DNA methylation editing. These results demonstrated a meaningful step forward toward realizing the specific editing of DNA methylation, suggesting the wide utility of our CNAMS for functional studies on epigenetic regulation and potential therapeutic strategies for related diseases.
A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice.
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.
Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice.
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.
Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse.
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.