Curated Optogenetic Publication Database

Abstract: The ability to modulate gene expression is crucial for studying gene function and programming cell behaviors. Combining the reliability of CRISPRi and the precision of optogenetics, the optoCRISPRi technique is emerging as an advanced tool for live-cell gene regulation. Since previous versions of optoCRISPRi often exhibit no more than a 10-fold dynamic range due to the leakage activity, they are not suitable for targets that are sensitive to such leakage or critical for cell growth. Here, we describe a green-light-activated CRISPRi system with a high dynamic range (40 fold) and the flexibility of changing targets in Escherichia coli. Our optoCRISPRi-HD system can efficiently repress essential genes, nonessential genes, or inhibit the initiation of DNA replication. Providing a regulative system with high resolution over space-time and extensive targets, our study would facilitate further research involving complex gene networks, metabolic flux redirection, or bioprinting.

Abstract: Retraction: "Long noncoding RNA ZFPM2-AS1 is involved in lung adenocarcinoma via miR-511-3p/AFF4 pathway," by Juan Li, Jun Ge, Ye Yang, Bin Liu, Min Zheng, and Rui Shi, J Cell Biochem. 2020; 2534-2542: The above article, published online on November 6, 2019, in Wiley Online Library (https://doi.org/10.1002/jcb.29476) has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed after the authors stated that unintentional errors occurred during the research process, and the experimental results cannot be verified. Thus, the conclusions are considered to be invalid. The authors were not available for a final confirmation of the retraction.

Abstract: Gene expression is inherently dynamic, due to complex regulation and stochastic biochemical events. However, the effects of these dynamics on cell phenotypes can be difficult to determine. Researchers have historically been limited to passive observations of natural dynamics, which can preclude studies of elusive and noisy cellular events where large amounts of data are required to reveal statistically significant effects. Here, using recent advances in the fields of machine learning and control theory, we train a deep neural network to accurately predict the response of an optogenetic system in Escherichia coli cells. We then use the network in a deep model predictive control framework to impose arbitrary and cell-specific gene expression dynamics on thousands of single cells in real time, applying the framework to generate complex time-varying patterns. We also showcase the framework’s ability to link expression patterns to dynamic functional outcomes by controlling expression of the tetA antibiotic resistance gene. This study highlights how deep learning-enabled feedback control can be used to tailor distributions of gene expression dynamics with high accuracy and throughput.

Abstract: Optogenetic actuators have revolutionized the resolution at which we can assert control over biological processes in living systems. In plants, deployment of optogenetics is challenging due to the need for these light-responsive systems to maintain a single activation state in conventional horticultural environments with light-dark cycling. Furthermore, many available optogenetic actuators are based on plant photoreceptors that might crosstalk with endogenous signaling processes, while others depend on exogenously supplied cofactors. To overcome such challenges, we have developed Highlighter; a synthetic, light-gated gene expression system tailored for in planta function. Highlighter is based on the photoswitchable CcaS-CcaR system from cyanobacteria and is repurposed for plants as a fully genetically encoded system, engineered to photoswitch with the endogenous plant chromophore, phytochromobilin. We deployed Highlighter in transiently transformed Nicotiana benthamiana for optogenetic control of fluorescent protein expression and innate immune responses. Using light to guide differential fluorescent protein expression in nuclei of neighboring cells, we demonstrate unprecedented spatiotemporal control of target gene expression. We furthermore regulate activation of plant immunity by modulating the spectral composition of white light, demonstrating optogenetic control of a biological process in horticultural light environments. Highlighter is a step forward for optogenetics in plants and a technology for high-resolution gene induction that will advance fundamental plant biology and provide new opportunities for crop improvement.

Abstract: Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits.

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: 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: 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: 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: Unraveling the transformative power of optogenetics in biology requires sophisticated engineering for the creation and optimization of light-regulatable proteins. In addition, diverse strategies have been used for the tuning of these light-sensitive regulators. This review highlights different protein engineering and synthetic biology approaches, which might aid in the development and optimization of novel optogenetic proteins (Opto-proteins). Focusing on non-neuronal optogenetics, chromophore availability, general strategies for creating light-controllable functions, modification of the photosensitive domains and their fusion to effector domains, as well as tuning concepts for Opto-proteins are discussed. Thus, this review shall not serve as an encyclopedic summary of light-sensitive regulators but aims at discussing important aspects for the engineering of light-controllable proteins through selected examples.

Abstract: Chemical induction is one of the most common modalities used to manipulate gene expression in living systems. However, chemical induction can be toxic or expensive that compromise the economic feasibility when it comes to industrial-scale synthetic biology applications. These complications have driven the pursuit of better induction systems. Optogenetics technique can be a solution as it not only enables dynamic control with unprecedented spatiotemporal precision but also is inexpensive and eco-friendlier. The optogenetic technique harnesses natural light-sensing modules that are genetically encodable and re-programmable in various hosts. By further engineering these modules to connect with the microbial regulatory machinery, gene expression and protein activity can be finely tuned simply through light irradiation. Recent works on applying optogenetics to microbial synthetic biology have yielded remarkable achievements. To further expand the usability of optogenetics, more optogenetic tools with greater portability that are compatible with different microbial hosts need to be developed. This review focuses on non-opsin optogenetic systems and the current state of optogenetic advancements in microbes, by showcasing the different designs and functions of optogenetic tools, followed by an insight into the optogenetic approaches used to circumvent challenges in synthetic biology.

Abstract: Optogenetics has been used in a variety of microbial engineering applications, such as chemical and protein production, studies of cell physiology, and engineered microbe-host interactions. These diverse applications benefit from the precise spatiotemporal control that light affords, as well as its tunability, reversibility, and orthogonality. This combination of unique capabilities has enabled a surge of studies in recent years investigating complex biological systems with completely new approaches. We briefly describe the optogenetic tools that have been developed for microbial engineering, emphasizing the scientific advancements that they have enabled. In particular, we focus on the unique benefits and applications of implementing optogenetic control, from bacterial therapeutics to cybergenetics. Finally, we discuss future research directions, with special attention given to the development of orthogonal multichromatic controls. With an abundance of advantages offered by optogenetics, the future is bright in microbial engineering. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 13 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Abstract: Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10-CcaS#10-CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10-CcaS#10-CcaR system was combined with a blue light-activated YF1-FixJ-PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene (gltA) and PHB synthesis gene (phbCAB) expression. Consequently, the strain RBS34, which has high gltA expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.

Abstract: Advances in genetic engineering, combined with the development of optical technologies, have allowed optogenetics to broaden its area of possible applications in recent years. However, the application of optogenetic tools in industry, including biotechnology and the production of biomaterials, is still limited, because each practical task requires the engineering of a specific optogenetic system. In this review, we discuss recent advances in the use of optogenetic tools in the production of biofuels and valuable chemicals, the synthesis of biomedical and polymer materials, and plant agrobiology. We also offer a comprehensive analysis of the properties and industrial applicability of light-controlled and other smart biomaterials. These data allow us to outline the prospects for the future use of optogenetics in bioindustry.

Abstract: Owing to its ubiquity and easy availability in nature, light has been widely employed to control complex cellular behaviors. Light-sensitive proteins are the foundation to such diverse and multilevel adaptive regulations in a large range of organisms. Due to their remarkable properties and potential applications in engineered systems, exploration and engineering of natural light-sensitive proteins have significantly contributed to expand optogenetic toolboxes with tailor-made performances in synthetic genetic circuits. Progressively, more complex systems have been designed in which multiple photoreceptors, each sensing its dedicated wavelength, are combined to simultaneously coordinate cellular responses in a single cell. In this review, we highlight recent works and challenges on multiplexed optogenetic circuits in natural and engineered systems for a dynamic regulation breakthrough in biotechnological applications.

Abstract: Optogenetics holds the promise of controlling biological processes with superb temporal and spatial resolution at minimal perturbation. Although many of the light-reactive proteins used in optogenetic systems are derived from prokaryotes, applications were largely limited to eukaryotes for a long time. In recent years, however, an increasing number of microbiologists use optogenetics as a powerful new tool to study and control key aspects of bacterial biology in a fast and often reversible manner. After a brief discussion of optogenetic principles, this review provides an overview of the rapidly growing number of optogenetic applications in bacteria, with a particular focus on studies venturing beyond transcriptional control. To guide future experiments, we highlight helpful tools, provide considerations for successful application of optogenetics in bacterial systems, and identify particular opportunities and challenges that arise when applying these approaches in bacteria.

Abstract: This review adds the bilin-binding phytochromes to the Chemical Reviews thematic issue "Optogenetics and Photopharmacology". The work is structured into two parts. We first outline the photochemistry of the covalently bound tetrapyrrole chromophore and summarize relevant spectroscopic, kinetic, biochemical, and physiological properties of the different families of phytochromes. Based on this knowledge, we then describe the engineering of phytochromes to further improve these chromoproteins as photoswitches and review their employment in an ever-growing number of different optogenetic applications. Most applications rely on the light-controlled complex formation between the plant photoreceptor PhyB and phytochrome-interacting factors (PIFs) or C-terminal light-regulated domains with enzymatic functions present in many bacterial and algal phytochromes. Phytochrome-based optogenetic tools are currently implemented in bacteria, yeast, plants, and animals to achieve light control of a wide range of biological activities. These cover the regulation of gene expression, protein transport into cell organelles, and the recruitment of phytochrome- or PIF-tagged proteins to membranes and other cellular compartments. This compilation illustrates the intrinsic advantages of phytochromes compared to other photoreceptor classes, e.g., their bidirectional dual-wavelength control enabling instant ON and OFF regulation. In particular, the long wavelength range of absorption and fluorescence within the "transparent window" makes phytochromes attractive for complex applications requiring deep tissue penetration or dual-wavelength control in combination with blue and UV light-sensing photoreceptors. In addition to the wide variability of applications employing natural and engineered phytochromes, we also discuss recent progress in the development of bilin-based fluorescent proteins.

Abstract: Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field.

Abstract: Optogenetics initially used plant photoreceptors to monitor neural circuits, later it has expanded to include engineered plant photoreceptors. Recently photoreceptors from bacteria, algae and cyanobacteria have been used as an optogenetic tool. Bilin-based photoreceptors are common light-sensitive photoswitches in plants, algae, bacteria and cyanobacteria. Here we discuss the photoreceptors from cyanobacteria. Several new photoreceptors have been explored in cyanobacteria which are now proposed as cyanobacteriochrome. The domains in the cyanobacteriochrome, light-induced signaling transduction, photoconversion, are the most attractive features for the optogenetic system. The wider spectral feature of cyanobacteriochrome from UV to visible radiation makes it a light potential sensitive optogenetic tool. Besides, cyanobacterial phytochrome responses to yellow, orange and blue light have more application in optogenetics. This chapter summarizes the photoconversion, phototaxis, cell aggregation, cell signaling mediated by cyanobacteriochrome and cyanophytochrome. As there is a wide range of cyanobacteriochrome and its combination delivers a varied light-sensitive response. Besides coordination among cyanobacteriochromes in cell signaling reduces the engineering of photoreceptors for the optogenetic system.

Abstract: Light has become established as a tool not only to visualize and investigate but also to steer biological systems. This review starts by discussing the unique features that make light such an effective control input in biology. It then gives an overview of how light‐control came to progress, starting with photoactivatable compounds and leading up to current genetic implementations using optogenetic approaches. The review then zooms in on optogenetics, focusing on photosensitive proteins, which form the basis for optogenetic engineering using synthetic biological approaches. As the regulation of transcription provides a highly versatile means for steering diverse biological functions, the focus of this review then shifts to transcriptional light regulators, which are presented in the biotechnologically highly relevant model organism Escherichia coli.

Abstract: Optimization of engineered biological systems requires precise control over the rates and timing of gene expression. Optogenetics is used to dynamically control gene expression as an alternative to conventional chemical-based methods since it provides a more convenient interface between digital control software and microbial culture. Here, we describe the construction of a real-time optogenetics platform, which performs closed-loop control over the CcaR-CcaS two-plasmid system in Escherichia coli. We showed the first model-based design approach by constructing a nonlinear representation of the CcaR-CcaS system, tuned the model through open-loop experimentation to capture the experimental behavior, and applied the model in silico to inform the necessary changes to build a closed-loop optogenetic control system. Our system periodically induces and represses the CcaR-CcaS system while recording optical density and fluorescence using image processing techniques. We highlight the facile nature of constructing our system and how our model-based design approach will potentially be used to model other systems requiring closed-loop optogenetic control.

Abstract: Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.

Abstract: Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.

Abstract: Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.