Curated Optogenetic Publication Database

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 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: Necroptosis plays a crucial role in the progression of various diseases and has gained substantial attention for its potential to activate antitumor immunity. However, the complex signaling networks that regulate necroptosis have made it challenging to fully understand its mechanisms and translate this knowledge into therapeutic applications. To address these challenges, an optogenetically activatable necroptosis system is developed that allows for precise spatiotemporal control of key necroptosis regulators, bypassing complex upstream signaling processes. The system, specifically featuring optoMLKL, demonstrates that it can rapidly assemble into functional higher-order "hotspots" within cellular membrane compartments, independent of RIPK3-mediated phosphorylation. Moreover, the functional module of optoMLKL significantly enhances innate immune responses by promoting the release of iDAMPs and cDAMPs, which are critical for initiating antitumor immunity. Furthermore, optoMLKL exhibits antitumor effects when activated in patient-derived pancreatic cancer organoids, highlighting its potential for clinical application. These findings will pave the way for innovative cancer therapies by leveraging optogenetic approaches to precisely control and enhance necroptosis.

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: The actin cytoskeleton and its nanoscale organization are central to all eukaryotic cells-powering diverse cellular functions including morphology, motility, and cell division-and is dysregulated in multiple diseases. Historically studied largely with purified proteins or in isolated cells, tools to study cell type-specific roles of actin in multicellular contexts are greatly needed. DeActs are recently created, first-in-class genetic tools for perturbing actin nanostructures and dynamics in specific cell types across diverse eukaryotic model organisms. Here, ChiActs are introduced, the next generation of actin-perturbing genetic tools that can be rapidly activated in cells and optogenetically targeted to distinct subcellular locations using light. ChiActs are composed of split halves of DeAct-SpvB, whose potent actin disassembly-promoting activity is restored by chemical-induced dimerization or allosteric switching. It is shown that ChiActs function to rapidly induce actin disassembly in several model cell types and are able to perturb actin-dependent nano-assembly and cellular functions, including inhibiting lamellipodial protrusions and membrane ruffling, remodeling mitochondrial morphology, and reorganizing chromatin by locally constraining actin disassembly to specific subcellular compartments. ChiActs thus expand the toolbox of genetically-encoded tools for perturbing actin in living cells, unlocking studies of the many roles of actin nano-assembly and dynamics in complex multicellular systems.

Abstract: The light chain of tetanus neurotoxin (TeNT) is a 52 kD metalloprotease that potently inhibits synaptic transmission by cleaving the endogenous vesicle fusion protein VAMP2. To mitigate the toxicity of TeNT and harness it as a conditional tool for neuroscience, we engineered Light-Activated TeNT (LATeNT) via insertion of the light-sensitive LOV domain into an allosteric site. LATeNT was optimized by directed evolution and shown to have undetectable activity in the dark mammalian brain. Following 30 seconds of weak blue light exposure, however, LATeNT potently inhibited synaptic transmission in multiple brain regions. The effect could be reversed over 24 hours. We used LATeNT to discover an interneuron population in hippocampus that controls anxiety-like behaviors in mouse, and to control the secretion of endogenous insulin from pancreatic beta cells. Synthetic circuits incorporating LATeNT converted drug, Ca2+, or receptor activation into transgene expression or reporter protein secretion. Due to its large dynamic range, rapid kinetics, and highly specific mechanism of action, LATeNT should be a robust tool for conditional proteolysis and spatiotemporal control of synaptic transmission in vivo.

Abstract: Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.

Abstract: Inducible protein switches are currently limited for use in tissues and organisms because common inducers cannot be controlled with precision in space and time in optically dense settings. Here, we introduce a protein that can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization using temperature) oligomerizes and translocates to the plasma membrane when temperature is lowered. We generated a library of Melt variants with switching temperatures ranging from 30 °C to 40 °C, including two that operate at and above 37 °C. Melt was a highly modular actuator of cell function, permitting thermal control over diverse processes including signaling, proteolysis, nuclear shuttling, cytoskeletal rearrangements and cell death. Finally, Melt permitted thermal control of cell death in a mouse model of human cancer. Melt represents a versatile thermogenetic module for straightforward, non-invasive and spatiotemporally defined control of mammalian cells with broad potential for biotechnology and biomedicine.

Abstract: Animal cells dismantle their nuclear envelope (NE) at the beginning and reconstruct it at the end of mitosis. This process is closely coordinated with spindle pole organization: poles enlarge at mitotic onset and reduce size as mitosis concludes. The significance of this coordination remains unknown. Here, we demonstrate that Aurora A maintains a pole-localized protein NuMA in a dynamic state during anaphase. Without Aurora A, NuMA shifts from a dynamic to a solid phase, abnormally accumulating at the poles, leading to chromosome bending and misshaped nuclei formation around poles. NuMA localization relies on interactions with dynein/dynactin, its coiled-coil domain, and intrinsically disordered region (IDR). Mutagenesis experiments revealed that cation-π interactions within IDR are key for NuMA localization, while glutamine residues trigger its solid-state transition upon Aurora A inhibition. This study emphasizes the role of the physical properties of spindle poles in organizing the nucleus and genome post-mitosis.

Abstract: Photosynthetic cyanobacteria can be utilised in biotechnology as environmentally sustainable cell factories to convert CO2 into a diverse range of biochemicals. However, a lack of molecular tools available for precise and dynamic control of gene expression hinders metabolic engineering and contributes to low product titres. Optogenetic tools enable light-regulated control of gene expression with high tunability and reversibility. To date, their application in cyanobacteria is limited and transferability between species remains unclear. In this study, we expressed the blue light-repressible YF1/FixJ and the green/red light-responsive CcaS/CcaR systems in Synechococcus sp. PCC 7002 and characterised their performance using GFP fluorescence assays and qRT-PCR. The YF1/FixJ system of non-cyanobacterial origin showed poor performance with a maximum dynamic range of 1.5-fold despite several steps to improve this. By contrast, the CcaS/CcaR system originating from the cyanobacterium Synechocystis sp. PCC 6803 responded well to light wavelengths and intensities, with a 6-fold increased protein fluorescence output observed after 30 min of green light. Monitoring GFP transcript levels allowed us to quantify the kinetics of transcriptional activation and deactivation and to test the effect of both multiple green/red and light/dark cycles on system performance. Finally, we increased CcaS/CcaR system activity under green light through targeted genetic modifications to the pCpcG2 output promoter. This study provides a detailed characterisation of the behaviour of the CcaS/CcaR system in Synechococcus sp. PCC 7002, as well as underlining the complexity of transferring optogenetic tools across species.

Abstract: Oscillatory dynamics and their modulation are crucial for cellular decision-making; however, analysing these dynamics remains challenging. Here, we present a tool that combines the light-activated adenylate cyclase mPAC with the cAMP biosensor Pink Flamindo, enabling precise manipulation and real-time monitoring of cAMP oscillation frequencies in Dictyostelium. High-frequency modulation of cAMP oscillations induced cell aggregation and multicellular formation, even at low cell densities, such as a few dozen cells. At the population level, chemotactic aggregation is driven by modulated frequency signals. Additionally, modulation of cAMP frequency significantly reduced the amplitude of the shuttling behaviour of the transcription factor GtaC, demonstrating low-pass filter characteristics capable of converting subtle oscillation changes, such as from 6 min to 4 min, into gene expression. These findings enhance our understanding of frequency-selective cellular decoding and its role in cellular signalling and development.

Abstract: Cerebral organoids pioneered in replicating complex brain tissue architectures in vitro, offering a vast potential for human disease modeling. They enable the in vitro study of human physiological and pathophysiological mechanisms of various neurological diseases and disorders. The trajectory of technological advancements in brain organoid generation and engineering over the past decade indicates that the technology might, in the future, mature into indispensable solutions at the horizon of personalized and regenerative medicine. In this review, we highlight recent advances in the engineering of brain organoids as disease models and discuss some of the challenges and opportunities for future research in this rapidly evolving field.

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: Among all photosynthetic life forms, cyanobacteria exclusively possess a water-soluble, light-sensitive carotenoprotein complex known as orange carotenoid proteins (OCPs), crucial for their photoprotective mechanisms. These protein complexes exhibit both structural and functional modularity, with distinct C-terminal (CTD) and N-terminal domains (NTD) serving as light-responsive sensor and effector regions, respectively. The majority of cyanobacterial genomes contain genes for OCP homologs and related proteins, highlighting their essential role in survival of the organism over time. Cyanobacterial photoprotection primarily involves the translocation of carotenoid entity into the NTD, leading to remarkable conformational changes in both domains and formation of metastable OCPR. Subsequently, OCPR interacts with phycobiliprotein, inducing the quenching of excitation energy and a significant reduction in PS II fluorescence yield. In dark conditions, OCPR detaches from phycobilisomes and reverts to OCPO in the presence of fluorescent recovery proteins (FRP), sustaining a continuous cycle. Research suggests that the modular structure of the OCPs, coupled with its unique light-driven dissociation and re-association capability, opens avenues for exploiting its potential as light-controlled switches, offering various biotechnological applications.

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: Komagataella phaffii, also known as Pichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1 promoter. Most K. phaffii bioprocesses for recombinant protein production rely on PAOX1 to achieve dynamic control in two-phase processes. Cells are first grown under conditions that repress PAOX1 (growth phase), followed by methanol-induced recombinant protein expression (production phase). In this study, we propose a methanol-free approach for dynamic metabolic control in K. phaffii using optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light-responsive transcription factor EL222 from Erythrobacter litoralis is used to regulate protein production from the PC120 promoter in K. phaffii with blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab-scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol-inducible bioprocesses using K. phaffii.

Abstract: Cells under high confinement form highly polarized hydrostatic pressure-driven, stable leader blebs that enable efficient migration in low adhesion, environments. Here we investigated the basis of the polarized bleb morphology of metastatic melanoma cells migrating in non-adhesive confinement. Using high-resolution time-lapse imaging and specific molecular perturbations, we found that EGF signaling via PI3K stabilizes and maintains a polarized leader bleb. Protein activity biosensors revealed a unique EGFR/PI3K activity gradient decreasing from rear-to-front, promoting PIP3 and Rac1-GTP accumulation at the bleb rear, with its antagonists PIP2 and RhoA-GTP concentrated at the bleb tip, opposite to the front-to-rear organization of these signaling modules in integrin-mediated mesenchymal migration. Optogenetic experiments showed that disrupting this gradient caused bleb retraction, underscoring the role of this signaling gradient in bleb stability. Mathematical modeling and experiments identified a mechanism where, as the bleb initiates, CD44 and ERM proteins restrict EGFR mobility in a membrane-apposed cortical actin meshwork in the bleb rear, establishing a rear-to-front EGFR-PI3K-Rac activity gradient. Thus, our study reveals the biophysical and molecular underpinnings of cell polarity in bleb-based migration of metastatic cells in non-adhesive confinement, and underscores how alternative spatial arrangements of migration signaling modules can mediate different migration modes according to the local microenvironment.

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: This volume explores the latest advancements in the field of optogenetics and how it uses cellular light-sensing components and genetic engineering to control proteins and biological processes. The book chapters are organized into four parts. Part One focuses on intracellular optogenetic components for control of specific cell functions; Part Two looks at externally supplied light regulators that do not require genetic manipulation of target cells; Part Three highlights optogenetic control of organelles, and Part Four introduces technical tools required for light induction in optogenetic experiments, as well as a method for performing and analyzing optogenetic cell-cell adhesion. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and practical, Optogenetics: Methods and Protocols is a valuable resource to help researchers understand and apply the concepts of optogenetics and the underlying bioengineering principles, and establish the required technical light-illumination setups for administering light inputs and analysis of experimental outcomes.

Abstract: The actin cytoskeleton forms a mesh-like network that drives cellular deformations. The network property is defined by the network density and the species of the actin-binding proteins. However, the relationship between the actin network density, the penetration ability of actin-binding proteins into the network, and resulting network dynamics remains elusive. Here, we report an in vitro optogenetic system, named OptoVCA, which induces Arp2/3-mediated actin network assembly on a lipid membrane. By changing the illumination power, duration, and pattern, the OptoVCA flexibly manipulates the density, thickness, and shape of the actin network. Taking these advantages, we examine the effects of the network density on the two representative actin-binding proteins, myosin and ADF/cofilin. We find that the penetration of myosin filaments into the network is strictly inhibited by only a several-fold increase in network density due to the steric hindrance. Furthermore, penetrated myosin filaments induce directional actin flow when the network has a density gradient. On the other hand, ADF/cofilin penetrates into the network regardless of network density, however, network disassembly is dramatically inhibited by only a several-fold increase in network density. Thus, the OptoVCA contributes to understanding cell mechanics through the examination of the network density-dependent effect on the actin-binding proteins.

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.