Curated Optogenetic Publication Database

Abstract: p53 protein, a crucial transcription factor in cellular responses to a wide variety of stress, regulates multiple target genes involved in tumor suppression, senescence induction, and metabolic functions. To characterize the context-dependent roles of p53, it is still needed to develop an experimental system that enables selective activation of p53 in cells and tissues. In this study, we developed an optogenetic tool, Opto-p53, to control p53 signaling by light. Opto-p53 was designed to trigger p53 signaling by reconstituting p53 N-terminal and C-terminal fragments with a light-inducible dimerization (LID) system. Upon light exposure, cells expressing Opto-p53 demonstrated p53 transcriptional activation, resulting in cell death and cell cycle arrest. We further enhanced the efficacy of light-induced p53 activation by introducing specific mutations into Opto-p53 fragments. Our findings unveil the capability of Opto-p53 to serve as a powerful tool for dissecting the complex roles of p53 in cellular processes, thereby contributing to the field of synthetic biology and providing general design principles for optogenetic tools using endogenous transcription factors.

Abstract: The Arabidopsis blue light photoreceptor cryptochrome 2 (CRY2) responds to blue light to initiate a variety of plant light-based behaviors and has been widely used for optogenetic engineering. Despite these important biological functions, the precise photoactivation mechanism of CRY2 remains incompletely understood. In light, CRY2 undergoes tetramerization and binds to partner proteins, including the transcription factor CIB1. Here we used yeast-two hybrid screening and deep mutational scanning to identify CRY2 amino acid changes that result in constitutive interaction with CIB1 in dark. The majority of CRY2 variants showing constitutive CIB1 interaction mapped to two regions, one near the FAD chromophore, and a second region located near the ATP binding site. Further testing of CRY2 variants from each region revealed three mapping near to the FAD binding pocket (D393S, D393A, and M378R) that also form constitutive CRY2-CRY2 homomers in dark, suggesting they adopt global conformational changes that mimic the photoactive state. Characterization of D393S in the homolog pCRY from Chlamydomonas reinhardtii using time-resolved UV-vis spectroscopy revealed that the FAD chromophore fails to form the neutral radical as signaling state upon illumination. Size exclusion chromatography of D393S shows the presence of homomers instead of a monomer in the dark, providing support for a hyperactive variant decoupled from the FAD. Our work provides new insight into photoactivation mechanisms of plant cryptochromes relevant for physiology and optogenetic application by revealing and localizing distinct activation pathways for light-driven CRY2-CIB1 and CRY2-CRY2 interactions.

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: Red light, characterized by superior tissue penetration and minimal phototoxicity, represents an ideal wavelength for optogenetic applications. However, the existing tools for reversible protein inhibition by red light remain limited. Here, we introduce R-LARIAT (red light-activated reversible inhibition by assembled trap), a novel optogenetic system enabling precise spatiotemporal control of protein function via 660 nm red-light-induced protein clustering. Our system harnesses the rapid and reversible binding of engineered light-dependent binders (LDBs) to the bacterial phytochrome DrBphP, which utilizes the endogenous mammalian biliverdin chromophore for red light absorption. By fusing LDBs with single-domain antibodies targeting epitope-tagged proteins (e.g., GFP), R-LARIAT enables the rapid sequestration of diverse proteins into light-responsive clusters. This approach demonstrates high light sensitivity, clustering efficiency, and sustained stability. As a proof of concept, R-LARIAT-mediated sequestration of tubulin inhibits cell cycle progression in HeLa cells. This system expands the optogenetic toolbox for studying dynamic biological processes with high spatial and temporal resolution and holds the potential for applications in living tissues.

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: Post-translational modifications (PTMs) are indispensable modulators of protein activity. Most cellular behaviors, from cell division to cytoskeletal organization, are controlled by PTMs, their misregulation being associated with a plethora of human diseases. Traditionally, the role of PTMs has been studied employing biochemical techniques. However, these approaches fall short when studying PTM dynamics in vivo. In recent years, functionalized protein binders have allowed the PTM of endogenous proteins by bringing an enzymatic domain in close proximity to the protein they recognize. To date, most of these methods lack the temporal control necessary to understand the complex effects triggered by PTMs. In this study, we have developed a method to phosphorylate endogenous Myosin in a light-inducible manner. The method relies both on nanobody-targeting and light-inducible activation in order to achieve both tight specificity and temporal control. We demonstrate that this technology is able to disrupt cytoskeletal dynamics during Drosophila embryonic development. Together, our results highlight the potential of combining optogenetics and protein binders for the study of the proteome in multicellular systems.

Abstract: Adrenoceptors (ARs) play a vital role in various physiological processes and are key therapeutic targets. The advent of optical control techniques, including optogenetics and photopharmacology, offers the potential to modulate AR signaling with precise temporal and spatial resolution. In this review, we summarize the latest advancements in the optical control of AR signaling, encompassing optogenetics, photocaged compounds, and photoswitchable compounds. We also discuss the limitations of current tools and provide an outlook on the next generation of optogenetic and photopharmacological tools. These emerging optical technologies not only enhance our understanding of AR signaling but also pave the way for potential therapeutic developments.

Abstract: Ferroptosis is a lytic, iron-dependent form of regulated cell death characterized by excessive lipid peroxidation and associated with necrosis spread in diseased tissues through unknown mechanisms. Using a novel optogenetic system for light-driven ferroptosis induction via degradation of the anti-ferroptotic protein GPX4, we show that lipid peroxidation and ferroptotic death can spread to neighboring cells through their closely adjacent plasma membranes. Ferroptosis propagation is dependent on cell distance and completely abolished by disruption of α-catenin-dependent intercellular contacts or by chelation of extracellular iron. Remarkably, bridging cells with a lipid bilayer or increasing contacts between neighboring cells enhances ferroptosis spread. Reconstitution of iron-dependent spread of lipid peroxidation between pure lipid, contacting liposomes provides evidence for the physicochemical mechanism involved. Our findings support a model in which iron-dependent lipid peroxidation propagates across proximal plasma membranes of neighboring cells, thereby promoting the transmission of ferroptotic cell death with consequences for pathological tissue necrosis spread.

Abstract: There is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats), coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here, we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs, BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa, and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo.

Abstract: Lipids are a major class of biological molecules, the primary components of cellular membranes, and critical signaling molecules that regulate cell biology and physiology. Due to their dynamic behavior within membranes, rapid transport between organelles, and complex and often redundant metabolic pathways, lipids have traditionally been considered among the most challenging biological molecules to study. In recent years, a plethora of tools bridging the chemistry-biology interface has emerged for studying different aspects of lipid biology. Here, we provide an overview of these approaches. We discuss methods for lipid detection, including genetically encoded biosensors, synthetic lipid analogs, and metabolic labeling probes. For targeted manipulation of lipids, we describe pharmacological agents and controllable enzymes, termed membrane editors, that harness optogenetics and chemogenetics. To conclude, we survey techniques for elucidating lipid-protein interactions, including photoaffinity labeling and proximity labeling. Collectively, these strategies are revealing new insights into the regulation, dynamics, and functions of lipids in cell biology.

Abstract: Calcium (Ca2+) release from intracellular stores, Ca2+ entry across the plasma membrane, and their coordination via store-operated Ca2+ entry (SOCE) are critical for receptor-activated Ca2+ oscillations. However, the precise mechanism of Ca2+ oscillations and whether their control loop resides at the plasma membrane or intracellularly remain unresolved. By examining the dynamics of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER)-localized Ca2+ sensor that activates the Orai1 channel on the plasma membrane for SOCE and in mast cells, we found that a significant proportion of cells exhibited STIM1 oscillations with the same periodicity as Ca2+ oscillations. These cortical oscillations, occurring in the cell's cortical region and shared with ER-plasma membrane (ER-PM) contact site proteins, were only detectable using total internal reflection fluorescence microscopy (TIRFM). Notably, STIM1 oscillations could occur independently of Ca2+ oscillations. Simultaneous imaging of cytoplasmic Ca2+ and ER Ca2+ with SEPIA-ER revealed that receptor activation does not deplete ER Ca2+, whereas receptor activation without extracellular Ca2+ influx induces cyclic ER Ca2+ depletion. However, under such nonphysiological conditions, cyclic ER Ca2+ oscillations lead to sustained STIM1 recruitment, indicating that oscillatory Ca2+ release is neither necessary nor sufficient for STIM1 oscillations. Using optogenetic tools to manipulate ER-PM contact site dynamics, we found that persistent ER-PM contact sites reduced the amplitude of Ca2+ oscillations without alteration of oscillation frequency. Together, these findings suggest an active cortical mechanism governs the rapid dissociation of ER-PM contact sites, thereby controlling the amplitude of oscillatory Ca2+ dynamics during receptor-induced Ca2+ oscillations.

Abstract: Light-based immunotherapy uses specific wavelengths of light to activate or modulate immune responses. It primarily employs two mechanisms: direct activation of immune cells and indirect modulation of the tumor microenvironment (TME). Several light-based technologies are under investigation or clinical use in immunotherapy, including photodynamic immunotherapy (PDIT) and photothermal therapy (PTT). Optogenetic tools have the potential to precisely control T-cell receptor activation, cytokine release, or the activity of other immune effector cells. Light-based technologies present innovative opportunities within the realm of immunotherapy. The ability to precisely regulate immune cell activation via optogenetics, alongside the improved targeting of cancer cells through photoimmunotherapy, signifies a transformative shift in our strategies for immune modulation. Although many of these technologies remain in the experimental stage for various applications, initial findings are encouraging, especially concerning cancer treatment and immune modulation. Continued research and clinical trials are essential to fully harness the capabilities of light technology in the context of immune cell therapy.

Abstract: Talin regulates the adhesion and migration of cells in part by promoting the affinity of integrins for extracellular matrix proteins, a process that in cells such as endothelial cells and platelets requires the direct interaction of talin with both the small GTPase Rap1 bound to GTP (Rap1-GTP) and the integrin β3 cytoplasmic tail. To study this process in more detail, we employed an optogenetic approach in living, immortalized endothelial cells to be able to regulate the interaction of talin with the plasma membrane. Previous studies identified talin as the Rap1-GTP effector for β3 integrin activation. Surprisingly, optogenetic recruitment of talin-1 (TLN1; herein referred to as talin) to the plasma membrane also led to the localized activation of Rap1 itself, apparently by talin competing for Rap1-GTP with SHANK3, a protein known to sequester Rap1-GTP and to block integrin activation. Rap1 activation by talin was localized to the cell periphery in suspension cells and within lamellipodia and pseudopodia in cells adherent to fibronectin. Thus, membrane-associated talin can play a dual role in regulating integrin function in endothelial cells: first, by releasing Rap1-GTP from its sequestration by SHANK3, and second, by serving as the relevant Rap1 effector for integrin 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 emerging technology that uses the light-responsive effects of photosensitive proteins to regulate the function of specific cells. This technique combines genetics with optics, allowing for the precise inhibition or activation of cell functions through the introduction of photosensitive proteins into target cells and subsequent light stimulation to activate these proteins. In recent years, numerous basic and clinical studies have demonstrated the unique advantages of this approach in the research and treatment of neurological disorders. This review aims to introduce the fundamental principles and techniques of optogenetics, as well as its applications in the research and treatment of neurological diseases.

Abstract: No organism is an island: organisms of varying taxonomic complexity, including genetic variants of a single species, can coexist in particular niches, cooperating for survival while simultaneously competing for environmental resources. In recent years, synthetic biology strategies have witnessed a surge of efforts focused on creating artificial microbial communities to tackle pressing questions about the complexity of natural systems and the interactions that underpin them. These engineered ecosystems depend on the number and nature of their members, allowing complex cell communication designs to recreate and create diverse interactions of interest. Due to its experimental simplicity, the budding yeast Saccharomyces cerevisiae has been harnessed to establish a mixture of varied cell populations with the potential to explore synthetic ecology, metabolic bioprocessing, biosensing, and pattern formation. Indeed, engineered yeast communities enable advanced molecule detection dynamics and logic operations. Here, we present a concise overview of the state-of-the-art, highlighting examples that exploit optogenetics to manipulate, through light stimulation, key yeast phenotypes at the community level, with unprecedented spatial and temporal regulation. Hence, we envision a bright future where the application of optogenetic approaches in synthetic communities (optoecology) illuminates the intricate dynamics of complex ecosystems and drives innovations in metabolic engineering strategies.

Abstract: G-protein-coupled receptors (GPCRs) are efficient guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G-protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GαGTP and free Gβγ and are major disease drivers. Evidence shows that the ambient low threshold signalling required for cells is likely supplemented by signalling regulators such as non-GPCR GEFs and guanine nucleotide dissociation inhibitors (GDIs). Activators of G-protein signalling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure-function relationship or signalling regulation in living cells. Here, we employed in silico structure-guided engineering of a novel optogenetic GDI, based on the AGS3's G-protein regulatory motif, to understand its GDI activity and induce standalone Gβγ signalling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases Gβγ, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signalling pathways and triggering GPCR-independent Gβγ signalling in cells and in vivo.

Abstract: Cells contain thousands of different lipids. Their rapid and redundant metabolism, dynamic movement, and many interactions with other biomolecules have justly earned lipids a reputation as a vexing class of molecules to understand. Further, as the cell’s hydrophobic metabolites, lipids assemble into supramolecular structures─most commonly bilayers, or membranes─from which they carry out myriad biological functions. Motivated by this daunting complexity, researchers across disciplines are bringing order to the seeming chaos of biological lipids and membranes. Here, we formalize these efforts as “synthetic lipid biology”. Inspired by the idea, central to synthetic biology, that our abilities to understand and build biological systems are intimately connected, we organize studies and approaches across numerous fields to create, manipulate, and analyze lipids and biomembranes. These include construction of lipids and membranes from scratch using chemical and chemoenzymatic synthesis, editing of pre-existing membranes using optogenetics and protein engineering, detection of lipid metabolism and transport using bioorthogonal chemistry, and probing of lipid–protein interactions and membrane biophysical properties. What emerges is a portrait of an incipient field where chemists, biologists, physicists, and engineers work together in proximity─like lipids themselves─to build a clearer description of the properties, behaviors, and functions of lipids and membranes.

Abstract: The regulation of PKC epsilon (PKCε) and its downstream effects is still not fully understood, making it challenging to develop targeted therapies or interventions. A more precise tool that enables spatiotemporal control of PKCε activity is thus required. Here, we describe a photo-activatable optogenetic PKCε probe (Opto-PKCε) consisting of an engineered PKCε catalytic domain and a blue-light inducible dimerization domain. Molecular dynamics and AlphaFold simulations enable rationalization of the dark-light activity of the optogenetic probe. We first characterize the binding partners of Opto-PKCε, which are similar to those of PKCε. Subsequent validation of the Opto-PKCε tool is performed with phosphoproteome analysis, which reveals that only PKCε substrates are phosphorylated upon light activation. Opto-PKCε could be engineered for recruitment to specific subcellular locations. Activation of Opto-PKCε in isolated hepatocytes reveals its sustained activation at the plasma membrane is required for its phosphorylation of the insulin receptor at Thr1160. In addition, Opto-PKCε recruitment to the mitochondria results in its lowering of the spare respiratory capacity through phosphorylation of complex I NDUFS4. These results demonstrate that Opto-PKCε may have broad applications for the studies of PKCε signaling with high specificity and spatiotemporal resolution.

Abstract: The balance of mitochondrial fission and fusion plays an important role in maintaining the stability of cellular homeostasis. Abnormal mitochondrial fission and fragmentation have been shown to be associated with oxidative stress, which causes a variety of human diseases from neurodegeneration disease to cancer. Therefore, the induction of mitochondrial aggregation and fusion may provide an alternative approach to alleviate these conditions. Here, an optogenetic-based mitochondrial aggregation system (Opto-MitoA) developed, which is based on the CRY2clust/CIBN light-sensitive module. Upon blue light illumination, CRY2clust relocates from the cytosol to mitochondria where it induces mitochondrial aggregation by CRY2clust homo-oligomerization and CRY2clust-CIBN hetero-dimerization. Our functional experiments demonstrate that Opto-MitoA-induced mitochondrial aggregation potently alleviates niclosamide-caused cell dysfunction in ATP production. This study establishes a novel optogenetic-based strategy to regulate mitochondrial dynamics in cells, which may provide a potential therapy for treating mitochondrial-related diseases.

Abstract: Live imaging techniques have revolutionized our understanding of paracrine signaling, a crucial form of cell-to-cell communication in biological processes. This review examines recent advances in visualizing and tracking paracrine factors through four key stages: secretion from producing cells, diffusion through extracellular space, binding to target cells, and activation of intracellular signaling within target cells. Paracrine factor secretion can be directly visualized by fluorescent protein tagging to ligand, or indirectly by visualizing the cleavage of the transmembrane pro-ligands or plasma membrane fusion of endosomes comprising the paracrine factors. Diffusion of paracrine factors has been studied using techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence decay after photoactivation (FDAP), and single-molecule tracking. Binding of paracrine factors to target cells has been visualized through various biosensors, including GPCR-activation-based (GRAB) sensors and Förster resonance energy transfer (FRET) probes for receptor tyrosine kinases. Finally, activation of intracellular signaling is monitored within the target cells by biosensors for second messengers, transcription factors, and so on. In addition to the imaging tools, the review also highlights emerging optogenetic and chemogenetic tools for triggering the release of paracrine factors, which is essential for associating the paracrine factor secretion to biological outcomes during the bioimaging of paracrine factor signaling.Key words: paracrine signaling, live imaging, biosensors, optogenetics, chemogenetics.

Abstract: Most bacteria lack membrane-enclosed organelles and rely on macromolecular scaffolds at different subcellular locations to recruit proteins for specific functions. Here, we demonstrate that the optogenetic CRY2-CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations with varying efficiencies in live Escherichia coli cells, including the nucleoid, the cell pole, the membrane, and the midcell division plane. Such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. We further show that CRY2-CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system. Finally, we test this optogenetic system in three additional bacterial species, Bacillus subtilis, Caulobacter crescentus, and Streptococcus pneumoniae, providing important considerations for this system's applicability in bacterial cell biology.

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: The CRISPR/Cas9 gene-editing technology, derived from the adaptive immune mechanisms of bacteria, has demonstrated remarkable advantages in fields such as gene function research and the treatment of genetic diseases due to its simplicity in design, precise targeting, and ease of use. Despite challenges such as off-target effects and cytotoxicity, effective spatiotemporal control strategies have been achieved for the CRISPR/Cas9 system through precise regulation of Cas9 protein activity as well as engineering of guide RNAs (gRNAs). This review provides a comprehensive analysis of the core components and functional mechanisms underlying the CRISPR/Cas9 system, highlights recent advancements in spatiotemporal control strategies, and discusses future directions for development.