Curated Optogenetic Publication Database

Abstract: Inducible expression of endogenous and foreign genes has been a pivotal driving force behind a lot many seminal breakthroughs in biotechnology. Synthetic biology, a very promising field, largely relies on transgene expression platforms which facilitate convenient and conditional regulation. Optogenetic approaches that exploit light to steer biological events, e.g., gene expression, with excellent spatiotemporal control, are often more precise compared to chemical induction. Light being an omnipresent environmental stimulus, serves as the ideal cue, and enables high spatiotemporal accuracy with respect to gene expression. In this review, we focus on different elements relevant to light-inducible gene expression - light-responsive promoters, light-regulated transcription factors, and photocaged inducers. Using light as a binary input function, we explore the essence of logic gates towards the development of gene expression circuits - thereby understanding the entanglement between optogenetics and synthetic biology. We primarily focus on prokaryotes, but also draw comparisons with analogous eukaryotic gene expression systems.

Abstract: The cytoskeleton is a dynamic intracellular network that governs cell shape, migration, division, and mechanotransduction. Precise spatiotemporal control of cytoskeletal regulation is essential for understanding how these processes are coordinated in physiology and disease, yet conventional pharmacological and genetic approaches often lack sufficient resolution or reversibility. Optogenetic technologies provide a powerful alternative by enabling light-controlled, noninvasive manipulation of cytoskeletal regulators with high temporal precision and subcellular specificity. This review summarizes recent advances in genetically encoded optogenetic tools for interrogating cytoskeletal dynamics. We discuss core design strategies, including allosteric regulation, light-induced oligomerization, heterodimerization, and dissociation, and highlight representative applications targeting actin filaments, microtubules, and upstream signaling pathways such as Rho family GTPases. We conclude by outlining current limitations and emerging directions, including improved tissue penetration, reduced phototoxicity, and multiplexed optical control, which are expected to further expand the utility of optogenetics in cytoskeleton research.

Abstract: Evolving dynamic, multi-state, and computational protein functionalities is challenging because it requires selection pressure on all the states of a protein of interest (POI) and the transitions between them. To create a continuous directed evolution paradigm for such properties, we genetically engineered budding yeast for optogenetic input to switch a POI "on" and "off," which, in turn, controls a Cdk1 cyclin that is essential for one cell-cycle stage but detrimental for another. The method, "optovolution," generates dynamic selection pressure on POI cycling at the timescale of tens of minutes. We used it to evolve 19 new variants of the LOV transcription factor El222, including in vivo green-light-responsive variants allowing LOV color-multiplexing. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing phycocyanobilin (PCB) unnecessary. Finally, we demonstrated the generality of the method by evolving a non-light-responsive AND gate (PEST-rtTA). Optovolution makes difficult-to-engineer protein functionalities continuously evolvable.

Abstract: As an interdisciplinary frontier integrating optical technologies and genetic principles, optogenetics enables precise spatiotemporal control of gene expression and neuronal activity via light-sensitive molecular assemblies, thereby driving transformative advancements in biomedical fields. Red/far-red light optogenetic tools, by virtue of the advantages of long wavelengths, have emerged as powerful platforms for deep-tissue manipulations for both basic researches and clinical applications. Although a number of in-depth studies on various red/far-red light optogenetic tools and their biomedical applications have been published, there has not yet been a comprehensive review that systematically summarizes the advancements of diverse researches on this type of optogenetics. This article systematically delineates the technology of red/far-red light optogenetics, focusing on the molecular mechanisms and biomedical applications of two core photoreceptor protein families: phytochromes and channelrhodopsins. Phytochromes distributed in plants, bacteria and fungi undergo reversible red/far-red light-driven conformational conversion, initiating downstream signaling cascades that support various optogenetic technologies. Channelrhodopsins, originally microalgal blue-light-gated cation channels, are engineered into red-shifted variants, enabling rapid and non-invasive red/far-red light-controlled neuronal excitability manipulation at precise spatiotemporal resolution. The representative case studies of applications of phytochromes-based optogenetic tools in gene editing, transcriptional regulation, light-gated drug delivery and deep tissue imaging and diagnosis; as well as applications of red-shifted channelrhodopsins-based optogenetic tools in spatiotemporally precise neuromodulation are discussed in detail. Moreover, the main technical challenges in the utilization of red/far-red light optogenetic tools are analyzed. With continuous advancements of wavelength-optimized actuators and closed-loop control architectures, red/far-red light optogenetic techniques are poised to drive multidisciplinary convergence, offering unprecedented tools for decoding cellular dynamics and accelerating therapeutic discoveries.

Abstract: Light is a pivotal environmental signal regulating diverse plant developmental and physiological processes, including seed germination, hypocotyl elongation, phototropism, metabolite biosynthesis, stress resistance, temperature response, and circadian rhythms. Multiple signal transduction pathways of ultraviolet, blue light, and red/far-red light as well as related protein interaction networks in plants have been identified. Deciphering the mechanisms of light perception and signal transduction is of great significance to crop breeding and optogenetic manipulation. Structural biology has profoundly advanced the studies of light signal transduction by elucidating high-resolution three-dimensional (3D) structures of photoreceptors and their downstream signaling components. These studies uncover the molecular basis underlying perception and transduction of different light signals by plants. This review summarizes key structural findings of plant light signal transduction, highlighting the architectures and molecular functions of photoreceptors and associated signaling factors. We also outline the mechanisms underlying photoreceptor activation, inhibition, and regulatory interactions within light signaling networks and discuss the challenges in this field.

Abstract: The light-mediated, specific, and precise control of cell functions enabled by optogenetics has become a versatile method for investigating and combatting cancer. An increasing set of optogenetic tools enables tightly controlled regulation of ion flux across biological membranes, gene expression, gene editing, and protein-protein interactions and is being used to interrogate hallmark traits of cancer at the cellular, subcellular, and organismic level. This enables, on the one hand, the identification of critical signaling circuits required for cancer development and progression in vitro and in animal models and can flag potential intervention points for pharmacologic interference. On the other hand, optogenetics can improve the level of control in cell-based therapeutics. The current article provides a review of optogenetic tools and approaches used in the cancer research field and their multiple applications for improving our understanding of signal transduction pathways, modulating immune functions in the tumor microenvironment, facilitating drug screening, or directly attacking cancer cells. Key advantages and achievements of optogenetics in the cancer research field and remaining barriers for clinical applications are discussed.

Abstract: Cellular signaling cascades rely on transfer of information from one protein to another or within a single protein. To facilitate signal integration, specific structural motifs evolved that allow signal processing and also enable modular downstream response integration, facilitating sophisticated regulatory mechanisms. On a structural level, especially coiled-coil helices are frequently observed as signaling motifs. In diguanylate cyclases (DGCs) featuring GGDEF domains, N-terminal coiled-coils frequently activate systems by rearrangements of the interdimer active site. The variety of sensory domains that modulate this structural equilibrium in response to different stimuli highlights the importance of DGCs in bacterial adaptation. One interesting example of sensor DGCs is blue light-activated light-oxygen-voltage (LOV)-GGDEF couples. Here, we describe molecular details of a two-stage mechanism that allows tight dark-state inhibition while enabling high enzymatic activities upon illumination, achieving fold changes exceeding 10,000-fold. Using an in vivo activity assay, we screened amino acid substitutions at the inhibitory interface and the sensor-effector linker region to identify variants that promote enzymatic activity in the dark. In combination with chimeras of LOV and GGDEF domains preventing inhibitory interface formation, we successfully stabilized elongated active-state conformations and confirmed the role of the inhibitory interface between sensor and effector in the tight dark-state inhibition. Interestingly, the initially generated chimeras are still light regulatable as long as the linker sequence is not stabilized in either inhibiting or stimulating coiled-coil register. Our results offer valuable insights for potential optogenetic applications but also demonstrate inherent challenges associated with Methylotenera sp. LOV-activated DGCs.

Abstract: The modification of key enzymes for chemical production plays a crucial role in enhancing the yield of targeted products. However, manipulating key nodes in specific signalling pathways remains constrained by traditional gene overexpression or knockout strategies. Discovering and designing optogenetic tools enable us to regulate enzymatic activity or gene expression at key nodes in a spatiotemporal manner, rather than relying solely on chemical induction throughout production processes. In this review, we discuss the recent applications of optogenetic tools in the regulation of microbial metabolites, plant sciences and disease therapies. We categorize optogenetic tools into five classes based on their distinct applications. First, light-induced gene expression schedules can balance the trade-off between chemical production and cell growth phases. Second, light-triggered liquid-liquid phase separation (LLPS) modules provide opportunities to co-localize and condense key enzymes for enhancing catalytic efficiency. Third, light-induced subcellular localized photoreceptors enable the relocation of protein of interest across various subcellular compartments, allowing for the investigation of their dynamic regulatory processes. Fourth, light-regulated enzymes can dynamically regulate production of cyclic nucleotides or investigate endogenous components similar with conditional depletion or recovery function of protein of interest. Fifth, light-gated ion channels and pumps can be utilized to investigate dynamic ion signalling cascades in both animals and plants, or to boost ATP accumulation for enhancing biomass or bioproduct yields in microorganisms. Overall, this review aims to provide a comprehensive overview of optogenetic strategies that have the potential to advance both basic research and bioindustry within the field of synthetic biology.

Abstract: Intracellular optogenetics represents a rapidly advancing biotechnology that enables precise, reversible control of protein activity, signaling dynamics, and cellular behaviours using genetically encoded, light-responsive systems. Originally pioneered in neuroscience through channelrhodopsins to manipulate neuronal excitability, the field has since expanded into diverse intracellular applications with broad implications for medicine, agriculture, and biomanufacturing. Key to these advances are photoreceptors such as cryptochrome 2 (CRY2), light-oxygen-voltage (LOV) domains, and phytochromes, which undergo conformational changes upon illumination to trigger conditional protein-protein interactions, localization shifts, or phase transitions. Recent engineering breakthroughs-including the creation of red-light responsive systems such as MagRed that exploit endogenous biliverdin-have enhanced tissue penetration, minimized phototoxicity, and expanded applicability to complex biological systems. This review provides an overarching synthesis of the molecular principles underlying intracellular optogenetic actuators, including the photophysical basis of light-induced conformational changes, oligomerization, and signaling control. We highlight strategies that employ domain fusions, rational mutagenesis, and synthetic circuits to extend their utility across biological and industrial contexts. We also critically assess current limitations, such as chromophore dependence, light delivery challenges, and safety considerations, so as to frame realistic paths towards translation. Looking ahead, future opportunities include multi-colour and multiplexed systems, integration with high-throughput omics and artificial intelligence, and development of non-invasive modalities suited for in vivo and industrial applications. Intracellular optogenetics is thus emerging as a versatile platform technology, with the potential to reshape how we interrogate biology and engineer cells for therapeutic, agricultural, and environmental solutions.

Abstract: Over the past two decades, optogenetics has evolved from a conceptual framework into a powerful and versatile technology for controlling cellular processes with light. Rooted in the discovery and characterization of natural photoreceptors, the field has advanced through the development of genetically encoded, light-sensitive proteins that enable precise spatiotemporal control of ion flux, intracellular signaling, gene expression, and protein interactions. This review traces key milestones in the emergence of optogenetics and highlights the development of major optogenetic tools. From the perspective of genetic tool innovation, the focus is on how these tools have been engineered and optimized for novel or enhanced functions, altered spectral properties, improved light sensitivity, subcellular targeting, and beyond. Their broadening applications are also explored across neuroscience, cardiovascular biology, hematology, plant sciences, and other emerging fields. In addition, current trends such as all-optical approaches, multiplexed control, and clinical translation, particularly in vision restoration are discussed. Finally, ongoing challenges are addressed and outline future directions in optogenetic tool development and in vivo applications, positioning optogenetics as a transformative platform for basic research and therapeutic advancement.

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) technology represents a landmark advance in the field of gene editing. However, conventional CRISPR/Cas systems are limited by inadequate temporal and spatial control. In recent years, the development of optically controlled CRISPR (Opto-CRISPR) technology has offered a novel solution to this issue. As a combination of optogenetics and the CRISPR technology, the Opto-CRISPR technology enables dynamic space-time-specific gene editing and regulation in cells and organisms. In this review, we concisely introduce the basic principles of Opto-CRISPR, summarize its operational mechanisms, and discuss its applications and recent advances across various research fields. In addition, this review analyzes the limitations of Opto-CRISPR, aiming to provide a reference for the development of this emerging field.

Abstract: Optogenetic bacterial technology is a cutting-edge approach that combines optogenetics and microbiology, offering a transformative strategy for disease diagnosis and therapy. This synergistic merger transcends the limitations of traditional diagnostic and therapeutic methodologies in a highly controllable, accurate and non-invasive manner. In this review, we introduce the optogenetic systems developed for microbial engineering and summarize fundamental in vitro design principles underlying light-responsive signal transduction in bacteria, as well as the optogenetic regulation of bacterial behaviors. We address multidisciplinary solutions to the challenges in the in vivo applications of light-controlled bacteria, such as limited light excitation, suboptimal delivery and targeting, and difficulties in signal tracking and management. Furthermore, we comprehensively highlight the recent progress in photo-responsive bacteria for disease diagnosis and therapy, and discuss how to accelerate translational applications.

Abstract: Plant science has entered a transformative era as genome editing enables precise DNA modifications to address global challenges such as climate adaptation and food security. These modifications are primarily driven by the integration of three modular components-DNA-targeting modules, effector modules, and control modules-that can be selectively activated or suppressed. The field has evolved from protein-based systems (e.g., zinc finger nucleases and transcription activator-like effector nucleases) to RNA-guided systems (e.g., CRISPR-Cas) that can control both genetic and epigenetic states. Modular pairing of DNA-targeting and effector domains, with or without inducible control, enables precise transcriptional regulation and chromatin remodeling. The present review examines these three modules and highlights strategies for their optimization. It also outlines innovative tools, such as optogenetic and receptor-integrated systems, that enable spatiotemporal control over genome editor expression. These modular approaches bypass traditional limitations and allow scientists to create plants with desirable traits, decipher complex gene networks, and promote sustainable agriculture.

Abstract: Neurodegenerative diseases involve toxic protein aggregation. Recent evidence suggests that biomolecular phase separation, a process in which proteins and nucleic acids form dynamic, liquid-like condensates, plays a key role in this aggregation. Optogenetics, originally developed to control neuronal activity with light, has emerged as a powerful tool to investigate phase separation in living systems. This is achieved by fusing disease-associated proteins to light-sensitive oligomerization domains, enabling researchers to induce or reverse condensate formation with precise spatial and temporal control. This review highlights how optogenetic systems such as OptoDroplet are being used to dissect the mechanisms of neurodegenerative disease. We examine how these tools have been applied in models of neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, and Huntington's disease. These studies implicate small oligomeric aggregates as key drivers of toxicity and highlight new opportunities for therapeutic screening. Finally, we discuss advances in light-controlled dissolution of condensates and future directions for applying optogenetics to combat neurodegeneration. By enabling precise, dynamic control of protein phase behavior in living systems, optogenetic approaches provide a powerful framework for elucidating disease mechanisms and informing the development of targeted therapies.

Abstract: Light is a critical environmental factor that governs the growth and development of plants. Plants have specialised photoreceptor proteins, which allow them to sense both quality and quantity of light and drive a wide range of responses critical for optimising growth, resource use and adaptation to changes in environment. Understanding the role of these photoreceptors in plant biology has opened up potential avenues for engineering crops with enhanced productivity by engineering photoreceptor activity and/or action. The ability to manipulate plant genomes through genetic engineering and synthetic biology approaches offers the potential to unlock new agricultural innovations by fine-tuning photoreceptors or photoreceptor pathways that control plant traits of agronomic significance. Additionally, optogenetic tools which allow for precise, light-triggered control of plant responses are emerging as powerful technologies for real-time manipulation of plant cellular responses. As these technologies continue to develop, the integration of photoreceptor engineering and optogenetics into crop breeding programs could potentially revolutionise how plant researchers tackle challenges of plant productivity. Here we provide an overview on the roles of key photoreceptors in regulating agronomically important traits, the current state of plant photoreceptor engineering, the emerging use of optogenetics and synthetic biology, and the practical considerations of applying these approaches to crop improvement. This review seeks to highlight both opportunities and challenges in harnessing photoreceptor engineering approaches for enhancing plant productivity. In this review, we provide an overview on the roles of key photoreceptors in regulating agronomically important traits, the current state of plant photoreceptor engineering, the emerging use of optogenetics and synthetic biology, and the practical considerations of applying these approaches to crop improvement.

Abstract: Bacterial therapy has emerged as a promising approach for disease treatment due to its environmental sensitivity, immunogenicity, and modifiability. However, the clinical application of engineered bacteria is limited by differences of expression levels in patients and possible off-targeting. Optogenetics, which combines optics and genetics, offers key advantages such as remote controllability, non-invasiveness, and precise spatiotemporal control. By utilizing optogenetic tools, the behavior of engineered bacteria can be finely regulated, enabling on-demand control of the dosage and location of their therapeutic products. In this review, we highlight the latest advancements in the optogenetic engineering of bacteria for light-controlled disease theranostics and therapeutic regulation. By constructing a three-dimensional analytical framework of “sense-produce-apply”, we begin by discussing the key components of bacterial optogenetic systems, categorizing them based on their photosensitive protein response to blue, green, and red light. Next, we introduce innovative light-producing tools that extend beyond traditional light sources. Then, special emphasis is placed on the biomedical applications of optogenetically engineered bacteria in treating diseases such as cancer, intestinal inflammation and systemic disease regulation. Finally, we address the challenges and future prospects of bacterial optogenetics, outlining potential directions for enhancing the safety and efficacy of light-controlled bacterial therapies. This review aims to provide insights and strategies for researchers working to advance the application of optogenetically engineered bacteria in drug delivery, precision medicine and therapeutic regulation.

Abstract: Transcription factors relay information from the external environment to gene regulatory networks that control cell physiology. To confer signalling specificity, robustness and coordination, these signalling networks use temporal communication codes, such as the amplitude, duration or frequency of signals. Although much is known about how temporal information is encoded, a mechanistic understanding of how gene regulatory networks decode signalling dynamics is lacking. Recent advances in our understanding of phase separation of transcriptional condensates provide new biophysical frameworks for both temporal encoding and decoding mechanisms. In this Perspective, we summarize the mechanisms by which transcriptional condensates could enable temporal decoding through signal adaptation, memory and persistence. We further outline methods to probe and manipulate dynamic communication codes of transcription factors and condensates to rationally control gene activation.

Abstract: Optogenetics has substantially enhanced our understanding of biological processes by enabling high-precision tracking and manipulation of individual cells. It relies on photosensitive proteins to monitor and control cellular activities, thereby paving the way for significant advancements in complex system research. Photosensitive proteins play a vital role in the development of optogenetics, facilitating the establishment of cutting-edge methods. Recent breakthroughs in protein design have opened up opportunities to develop protein-based tools that can precisely manipulate and monitor cellular activities. These advancements will significantly accelerate the development and application of optogenetic tools. This article emphasizes the pivotal role of protein design in the development of optogenetic tools, offering insights into potential future directions. We begin by providing an introduction to the historical development and fundamental principles of optogenetics, followed by an exploration of the operational mechanisms of key photosensitive domains, which includes clarifying the conformational changes they undergo in response to light, such as allosteric modulation and dimerization processes. Building on this foundation, we reveal the development of protein design tools that will enable the creation of even more sophisticated optogenetic techniques.

Abstract: Optogenetics is an empowering technology that uses light-responsive proteins to control biological processes. Because of its genetic tractability, abundance of genetic tools, and robust culturing conditions, Saccharomyces cerevisiae has served for many years as an ideal platform in which to study, develop, and apply a wide range of optogenetic systems. In many instances, yeast has been used as a steppingstone in which to characterize and optimize optogenetic tools to later be deployed in higher eukaryotes. More recently, however, optogenetic tools have been developed and deployed in yeast specifically for biotechnological applications, including in nonconventional yeasts. In this review, we summarize various optogenetic systems responding to different wavelengths of light that have been demonstrated in diverse yeast species. We then describe various applications of these optogenetic tools in yeast, particularly in metabolic engineering and recombinant protein production. Finally, we discuss emerging applications in yeast cybergenetics-the interfacing of yeast and computers for closed-loop controls of yeast bioprocesses-and the potential impact of optogenetics in other future biotechnological applications.

Abstract: Collective cell migration and tissue morphogenesis play a variety of important roles in the development of many species. Tissue morphogenesis often generates mechanical forces that alter cell shapes and arrangements, resembling collective cell migration-like behaviors. Genetic methods have been widely used to study collective cell migration and its like behavior, advancing our understanding of these processes during development. However, a growing body of research shows that collective cell migration during development is not a simple behavior but is often combined with other cellular and tissue processes. In addition, different surrounding environments can also influence migrating cells, further complicating collective cell migration during development. Due to the complexity of developmental processes and tissues, traditional genetic approaches often encounter challenges and limitations. Thus, some methods with spatiotemporal control become urgent in dissecting collective cell migration and tissue morphogenesis during development. Optogenetics is a method that combines optics and genetics, providing a perfect strategy for spatiotemporally controlling corresponding protein activity in subcellular, cellular or tissue levels. In this review, we introduce the basic mechanisms underlying different optogenetic tools. Then, we demonstrate how optogenetic methods have been applied in vivo to dissect collective cell migration and tissue morphogenesis during development. Additionally, we describe some promising optogenetic approaches for advancing this field. Together, this review will guide and facilitate future studies of collective cell migration in vivo and tissue morphogenesis by optogenetics.

Abstract: As synthetic biology advances, the necessity for robust biocontainment strategies for genetically engineered organisms (GEOs) grows increasingly critical to mitigate biosafety risks related to their potential environmental release. This paper aims to evaluate environment signal-dependent biocontainment systems for engineered organisms, focusing specifically on leveraging triggered responses and combinatorial systems. There are different types of triggers—chemical, light, temperature, and pH—this review illustrates how these systems can be designed to respond to environmental signals, ensuring a higher safety profile. It also focuses on combinatorial biocontainment to avoid consequences of unintended GEO release into an external environment. Case studies are discussed to demonstrate the practical applications of these systems in real-world scenarios.

Abstract: Plant phytochromes perceive red and far-red light to elicit adaptations to the changing environment. Downstream physiological responses revolve around red-light-induced interactions with phytochrome-interacting factors (PIF). Phytochromes double as thermoreceptors, owing to the pronounced temperature dependence of thermal reversion from the light-adapted Pfr to the dark-adapted Pr state. Here, we assess whether thermoreception may extend to the phytochrome:PIF interactions. While the association between Arabidopsis (Arabidopsis thaliana) PHYTOCHROME B (PhyB) and several PHYTOCHROME-INTERACTING FACTOR (PIF) variants moderately accelerates with temperature, the dissociation does more so, thus causing net destabilization of the phytochrome:PIF complex. Markedly different temperature profiles of PIF3 and PIF6 might underlie stratified temperature responses in plants. Accidentally, we identify a photoreception mechanism under strong continuous light, where the extent of phytochrome:PIF complexation decreases with red-light intensity rather than increases. Mathematical modeling rationalizes this attenuation mechanism and ties it to rapid red-light-driven Pr⇄Pfr interconversion and complex dissociation out of Pr. Varying phytochrome abundance, e.g., during diurnal and developmental cycles, and interaction dynamics, e.g., across different PIFs, modify the nature and extent of attenuation, thus permitting light-response profiles more malleable than possible for the phytochrome Pr⇄Pfr interconversion alone. Our data and analyses reveal a photoreception mechanism with implications for plant physiology, optogenetics, and biotechnological applications.

Abstract: Phytochromes are photoreceptor proteins in plants, fungi, and bacteria. They can adopt two photochromic states with differential biochemical responses. The structural changes transducing the signal from the chromophore to the biochemical output modules are poorly understood due to challenges in capturing structures of the dynamic, full-length protein. Here, we present cryoelectron microscopy (cryo-EM) structures of the phytochrome from Pseudomonas aeruginosa (PaBphP) in its resting (Pfr) and photoactivated (Pr) state. The kinase-active Pr state has an asymmetric, dimeric structure, whereas the kinase-inactive Pfr state opens up. This behavior is different from other known phytochromes and we explain it with the unusually short connection between the photosensory and output modules. Multiple sequence alignment of this region suggests evolutionary optimization for different modes of signal transduction in sensor proteins. The results establish a new mechanism for light-sensing by phytochrome histidine kinases and provide input for the design of optogenetic phytochrome variants.

Abstract: Far-red cyanobacteriochromes (CBCRs) are bilin-based photosensory proteins that promise to be novel optical agents in optogenetics and deep tissue imaging. Recent structural studies of a far-red CBCR 2551g3 have revealed a unique all-Z,syn chromophore conformation in the far-red-absorbing Pfr state. Understanding the photoswitching mechanism through bilin photoisomerization is important for developing novel biomedical applications. Here, we employ femtosecond spectroscopy and site-directed mutagenesis to systematically characterize the dynamics of wild-type 2551g3 and four critical mutants in the 15Z Pfr state. We captured local relaxations in several picoseconds and isomerization dynamics in hundreds of picoseconds. Most mutants exhibited faster local relaxation, while their twisting dynamics and photoproducts depend on specific protein-chromophore interactions around the D-ring and C-ring. These results collectively reveal a unique dynamic pattern of excited-state evolution arising from a relatively rigid protein environment, thereby elucidating the molecular mechanism of Pfr-state photoisomerization in far-red CBCRs.

Abstract: Synthetic Sc2.0 yeast strains contain hundreds to thousands of loxPsym recombination sites that allow restructuring of the Saccharomyces cerevisiae genome by SCRaMbLE. Thus, a highly diverse yeast population can arise from a single genotype. The selection of genetically diverse candidates with rearranged synthetic chromosomes for downstream analysis requires an efficient and straightforward workflow. Here we present loxTags, a set of qPCR primers for genotyping across loxPsym sites to detect not only deletions but also inversions and translocations after SCRaMbLE. To cope with the large number of amplicons, we generated qTagGer, a qPCR genotyping primer prediction tool. Using loxTag-based genotyping and long-read sequencing, we show that light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or a circular version of synthetic chromosome III.