Curated Optogenetic Publication Database

Abstract: Cell-surface receptors mediate communication between cells and their environment. Lateral membrane organization and dynamic receptor cluster formation are fundamental in signal transduction and cell signaling. However, it is not yet fully understood how receptor clustering modulates a wide variety of physiologically relevant processes. Recent growing evidence indicates that biological responses triggered by membrane receptors can be modulated even in the absence of the natural receptor ligand. We review the most recent findings on how ligand-independent receptor clustering can regulate transmembrane signaling. We discuss the latest technologies to control receptor assembly, such as DNA nanotechnology, optogenetics, and optochemistry, focusing on the biological relevance and unraveling of ligand-independent signaling.

Abstract: In recent years, light-responsive systems from the field of optogenetics have been applied to several areas of metabolic engineering with remarkable success. By taking advantage of light's high tunability, reversibility, and orthogonality to host endogenous processes, optogenetic systems have enabled unprecedented dynamical controls of microbial fermentations for chemical production, metabolic flux analysis, and population compositions in co-cultures. In this article, we share our opinions on the current state of this new field of metabolic optogenetics.We make the case that it will continue to impact metabolic engineering in increasingly new directions, with the potential to challenge existing paradigms for metabolic pathway and strain optimization as well as bioreactor operation.

Abstract: In optogenetics, as in nature, sensory photoreceptors serve to control cellular processes by light. Bacteriophytochrome (BphP) photoreceptors sense red and far-red light via a biliverdin chromophore and, in response, cycle between the spectroscopically, structurally, and functionally distinct Pr and Pfr states. BphPs commonly belong to two-component systems that control the phosphorylation of cognate response regulators and downstream gene expression through histidine kinase modules. We recently demonstrated that the paradigm BphP from Deinococcus radiodurans exclusively acts as a phosphatase but that its photosensory module can control the histidine kinase activity of homologous receptors. Here, we apply this insight to reprogram two widely used setups for bacterial gene expression from blue-light to red-light control. The resultant pREDusk and pREDawn systems allow gene expression to be regulated down and up, respectively, uniformly under red light by 100-fold or more. Both setups are realized as portable, single plasmids that encode all necessary components including the biliverdin-producing machinery. The triggering by red light affords high spatial resolution down to the single-cell level. As pREDusk and pREDawn respond sensitively to red light, they support multiplexing with optogenetic systems sensitive to other light colors. Owing to the superior tissue penetration of red light, the pREDawn system can be triggered at therapeutically safe light intensities through material layers, replicating the optical properties of the skin and skull. Given these advantages, pREDusk and pREDawn enable red-light-regulated expression for diverse use cases in bacteria.

Abstract: Molecular optogenetics is a highly dynamic research field. In the past two years, the field was characterized by the development of new allosteric switches as well as the forward integration of optogenetics research towards application. Further, two areas of research have significantly gathered momentum, the use of optogenetics to control liquid-liquid phase separation as well as the application of optogenetic tools in the extracellular space. Here, we review these areas and discuss future directions.

Abstract: Optogenetic approaches enable light-mediated control of cellular activities using genetically encoded photoreceptors. While optogenetic technology is already well established in neuroscience and fundamental research, the implementation of optogenetic tools in bacteriology is still emerging. Engineered bacteria with the specific optogenetic system that function at the transcriptional or post-translational level can sense and respond to light, allowing optogenetic control of bacterial behaviors. In this review, we give a brief overview of available optogenetic systems, including their mode of action, classification, and engineering strategies, and focus on optogenetic control of bacterial behaviors with the highlight of strategies for use of optogenetic systems.

Abstract: The H2 + H2 system has long been considered a benchmark system for ro-vibrational energy transfer in bimolecular collisions. However, most studies thus far have focused on collisions involving H2 molecules in the ground vibrational level or in the first excited vibrational state. While H2 + H2/HD collisions have received wide attention due to the important role they play in astrophysics, D2 + D2 collisions have received much less attention. Recently, Zhou et al. [ Nat. Chem. 2022, 14, 658-663, DOI: 10.1038/s41557-022-00926-z] examined stereodynamic aspects of rotational energy transfer in collisions of two aligned D2 molecules prepared in the v = 2 vibrational level and j = 2 rotational level. Here, we report quantum calculations of rotational and vibrational energy transfer in collisions of two D2 molecules prepared in vibrational levels up to v = 2 and identify key resonance features that contribute to the angular distribution in the experimental results of Zhou et al. The quantum scattering calculations were performed in full dimensionality and using the rigid-rotor approximation using a recently developed highly accurate six-dimensional potential energy surface for the H4 system that allows descriptions of collisions involving highly vibrationally excited H2 and its isotopologues.

Abstract: Optogenetic modalities as well as optochemical and photopharmacological strategies, collectively termed optical methods, have revolutionized the control of cellular functions via light with great spatiotemporal precision. In comparison to the major advances in the photomodulation of signaling activities noted in neuroscience, similar applications to endocrine cells of the pancreas, particularly insulin-producing β-cells, have been limited. The availability of tools allowing light-mediated changes in the trafficking of ions such as K+ and Ca2+ and signaling intermediates such as cyclic adenosine monophosphate (cAMP), renders β-cells and their glucose-stimulated insulin secretion (GSIS) amenable to optoengineering for drug-free control of blood sugar.

Abstract: Since the successful introduction of exogenous photosensitive proteins, channelrhodopsin, to neurons, optogenetics has enabled substantial understanding of profound brain function by selectively manipulating neural circuits. In an optogenetic system, optical stimulation can be precisely delivered to brain tissue to achieve regulation of cellular electrical activity with unprecedented spatio-temporal resolution in living organisms. In recent years, the development of various optical actuators and novel light-delivery techniques has greatly expanded the scope of optogenetics, enabling the control of other signal pathways in non-neuronal cells for different biomedical applications, such as phototherapy and immunotherapy. This review focuses on the recent advances in optogenetic regulation of cellular activities for photomedicine. We discuss emerging optogenetic tools and light-delivery platforms, along with a survey of optogenetic execution in mammalian and microbial cells.

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: To understand cell biological processes, like signalling pathways, protein movements, or metabolic processes, precise tools for manipulation are desired. Optogenetics allows to control cellular processes by light and can be applied at a high temporal and spatial resolution. In the last three decades, various optogenetic applications have been developed for animal, fungal, and prokaryotic cells. However, using optogenetics in plants has been difficult due to biological and technical issues, like missing cofactors, the presence of endogenous photoreceptors, or the necessity of light for photosynthesis, which potentially activates optogenetic tools constitutively. Recently developed tools overcome these limitations, making the application of optogenetics feasible also in plants. Here, we highlight the most useful recent applications in plants and give a perspective for future optogenetic approaches in plants science.

Abstract: Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200-1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research.

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: We review fundamental mechanisms and applications of OptoGels: hydrogels with light-programmable properties endowed by photoswitchable proteins ("optoproteins") found in nature. Light, as the primary source of energy on earth, has driven evolution to develop highly-tuned functionalities, such as phototropism and circadian entrainment. These functions are mediated through a growing family of optoproteins that respond to the entire visible spectrum ranging from ultraviolet to infrared by changing their structure to transmit signals inside of cells. In a recent series of articles, engineers and biochemists have incorporated optoproteins into a variety of extracellular systems, endowing them with photocontrollability. While other routes exist for dynamically controlling material properties, light-sensitive proteins have several distinct advantages, including precise spatiotemporal control, reversibility, substrate selectivity, as well as biodegradability and biocompatibility. Available conjugation chemistries endow OptoGels with a combinatorially large design space determined by the set of optoproteins and polymer networks. These combinations result in a variety of tunable material properties. Despite their potential, relatively little of the OptoGel design space has been explored. Here, we aim to summarize innovations in this emerging field and highlight potential future applications of these next generation materials. OptoGels show great promise in applications ranging from mechanobiology, to 3D cell and organoid engineering, and programmable cell eluting materials.

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.

Abstract: Light and temperature in plants are perceived by a common receptor, phytochrome B (phyB). How phyB distinguishes these signals remains elusive. Here, we report that phyB spontaneously undergoes phase separation to assemble liquid-like droplets. This capacity is driven by its C terminus through self-association, whereas the intrinsically disordered N-terminal extension (NTE) functions as a biophysical modulator of phase separation. Light exposure triggers a conformational change to subsequently alter phyB condensate assembly, while temperature sensation is directly mediated by the NTE to modulate the phase behavior of phyB droplets. Multiple signaling components are selectively incorporated into phyB droplets to form concentrated microreactors, allowing switch-like control of phyB signaling activity through phase transitions. Therefore, light and temperature cues are separately read out by phyB via allosteric changes and spontaneous phase separation, respectively. We provide a conceptual framework showing how the distinct but highly correlated physical signals are interpreted and sorted by one receptor.

Abstract: 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.

Abstract: Gene and cell therapies are widely recognized as future cancer therapeutics but poor controllability limits their clinical applications. Optogenetics, the use of light-controlled proteins to precisely spatiotemporally regulate the activity of genes and cells, opens up new possibilities for cancer treatment. Light of specific wavelength can activate the immune response, oncolytic activity and modulate cell signaling in tumor cells non-invasively, in dosed manner, with tissue confined action and without side effects of conventional therapies. Here, we review optogenetic approaches in cancer research, their clinical potential and challenges of incorporating optogenetics in cancer therapy. We critically discuss beneficial combinations of optogenetic technologies with therapeutic nanobodies, T-cell activation and CAR-T cell approaches, genome editors and oncolytic viruses. We consider viral vectors and nanoparticles for delivering optogenetic payloads and activating light to tumors. Finally, we highlight herein the prospects for integrating optogenetics into immunotherapy as a novel, fast, reversible and safe approach to cancer treatment.

Abstract: Harnessing the potential of optogenetics in biology requires methodologies from different disciplines ranging from biology, to mechatronics engineering, to control engineering. Light stimulation of a synthetic optogenetic construct in a given biological species can only be achieved via a suitable light stimulation platform. Emerging optogenetic applications entail a consistent, reproducible, and regulated delivery of light adapted to the application requirement. In this review, we explore the evolution of light-induction hardware-software platforms from simple illumination set-ups to sophisticated microscopy, microtiter plate and bioreactor designs, and discuss their respective advantages and disadvantages. Here, we examine design approaches followed in performing optogenetic experiments spanning different cell types and culture volumes, with induction capabilities ranging from single cell stimulation to entire cell culture illumination. The development of automated measurement and stimulation schemes on these platforms has enabled researchers to implement various in silico feedback control strategies to achieve computer-controlled living systems-a theme we briefly discuss in the last part of this review.

Abstract: The OptoAAV technology allows spatially defined delivery of transgenes into native target cells down to single-cell resolution by the illumination with cell-compatible and tissue-penetrating red light. The system is based on an adeno-associated viral (AAV) vector of serotype 2 with an engineered capsid (OptoAAV) and a photoreceptor-containing adapter protein mediating the interaction of the OptoAAV with the surface of the target cell in response to low doses of red and far-red light. In this article, we first provide detailed protocols for the production, purification, and analysis of the OptoAAV and the adapter protein. Afterward, we describe in detail the application of the OptoAAV system for the light-controlled transduction of human cells with global and patterned illumination. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production, purification, and analysis of PhyB-DARPinEGFR adapter protein Basic Protocol 2: Production, purification, and analysis of OptoAAV Basic Protocol 3: Red light-controlled viral transduction with the OptoAAV system Support Protocol: Spatially resolved transduction of two transgenes with the OptoAAV system.

Abstract: Synthetic biology is a field of research in which molecular parts (mostly nucleic acids and proteins) are de novo created or modified and then used either alone or in combination to achieve new functions that can help solve the problems of our modern society. In synthetic microbiology, microbes are employed rather than other organisms or cell-free systems. Optogenetics, a relatively recently established technology that relies on the use of genetically encoded photosensitive proteins to control biological processes with high spatiotemporal precision, offers the possibility to empower synthetic (micro)biology applications due to the many positive features that light has as an external trigger. In this review, we describe recent synthetic microbiology applications that made use of optogenetics after briefly introducing the molecular mechanism behind some of the most employed optogenetic tools. We highlight the power and versatility of this technique, which opens up new horizons for both research and industry.

Abstract: A comprehensive understanding of signaling mechanisms helps interpret fundamental biological processes and restore cell behavior from pathological conditions. Signaling outcome depends not only on the activity of each signaling component but also on their dynamic interaction in time and space, which remains challenging to probe by biochemical and cell-based assays. Opsin-based optogenetics has transformed neural science research with its spatiotemporal modulation of the activity of excitable cells. Motivated by this advantage, opsin-free optogenetics extends the power of light to a larger spectrum of signaling molecules. This review summarizes commonly used opsin-free optogenetic strategies, presents a historical overview of split protein complementation, and highlights the adaptation of split protein recombination as optogenetic sensors and actuators.

Abstract: Oral diseases such as gingivitis, periodontitis, and oral cancer affect millions of people worldwide. Much research has been conducted to understand the pathogenetic mechanisms of these diseases and translate this knowledge into therapeutics. This review aims to take the reader on a journey from the initial molecular discoveries to complex regenerative issues in oral medicine. For this, a semi-systematic literature search was carried out in Medline and Web of Science databases to retrieve the primary literature describing oral cell models and biomaterial applications in oral regenerative medicine. First, an in vitro cell model of gingival keratinocytes is discussed, which illustrates patho- and physiologic principles in the context of oral epithelial homeostasis and carcinogenesis and represents a cellular tool to understand biomaterial-based approaches for periodontal tissue regeneration. Consequently, a layered gradient nonwoven (LGN) is described, which demonstrates that the key features of biomaterials serve as candidates for oral tissue regeneration. LGN supports proper tissue formation and obeys the important principles for molecular mechanotransduction. Furthermore, current biomaterial-based tissue regeneration trends, including polymer modifications, cell-based treatments, antimicrobial peptides and optogenetics, are introduced to represent the full spectrum of current approaches to oral disease mitigation and prevention. Altogether, this review is a foray through established and new concepts in oral regenerative medicine and illustrates the process of knowledge translation from basic molecular and cell biological research to future clinical applications.

Abstract: Early embryogenesis requires rapid division of pluripotent blastomeres, regulated genome activation, precise spatiotemporal signaling to pattern cell fate, and morphogenesis to shape primitive tissue architectures. The complexity of this process has inspired researchers to move beyond simple genetic perturbation into engineered devices and synthetic biology tools to permit temporal and spatial manipulation of the control systems guiding development. By precise alteration of embryo organization, it is now possible to advance beyond basic analytical strategies and directly test the sufficiency of models for developmental regulation. Separately, advances in micropatterning and embryoid culture have facilitated the bottom-up construction of complex embryo tissues allowing ex vivo systems to recapitulate even later stages of development. Embryos fertilized and grown ex vivo offer an excellent opportunity to exogenously perturb fundamental pathways governing embryogenesis. Here we review the technologies developed to thermally modulate the embryo cell cycle, and optically regulate morphogen and signaling pathways in space and time, specifically in the blastula embryo. Additionally, we highlight recent advances in cell patterning in two and three dimensions that have helped reveal the self-organizing properties and gene regulatory networks guiding early embryo organization.

Abstract: Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

Abstract: In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light-responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome-based hydrogels with an increased dynamic range for the storage modulus but a different light-response for the loss modulus compared to the original Cph1-based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1-R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems.