Curated Optogenetic Publication Database

Abstract: Glucose is an essential energy source in living cells and is involved in various phenomena. To understand the roles of glucose, measuring cellular glucose levels is important. Here, we developed a bioluminescent glucose indicator called LOTUS-Glc. Unlike fluorescence, bioluminescence doesn't require excitation light when imaging. Using LOTUS-Glc, we demonstrated drug effect evaluation, concurrent use with the optogenetic tool in HEK293T cells, and the measurement of light-dependent glucose fluctuations in plant-derived protoplasts. LOTUS-Glc would be a useful tool for understanding the roles of glucose in living organisms.

Abstract: Morphogen gradients convey essential spatial information during tissue patterning. While both concentration and timing of morphogen exposure are crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homologue of NF-κB, which orchestrates dorso-ventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning, and characterized the resulting effect on spatio-temporal transcription of target genes in terms of timing, coordination, and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation (sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL is detectable at the level of single cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally-resolved and reversible modulation of a morphogen in vivo, combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.

Abstract: Post-translational modifications (PTMs) are indispensable modulators of protein activity. Most cellular behaviours, from cell division to cytoskeletal organization, are controlled by PTMs, their miss-regulation 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 post-translational modification 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: CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins, natural inhibitors of CRISPR-Cas systems, provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and -Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains of human origin, that together respond to six different, clinically-relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and -C CRISPR-Cas9s, and -Cas12a. In sum, this work establishes a versatile platform for multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology and potential for therapeutic use in the future.

Abstract: Light-Oxygen-Voltage (LOV) domains are the protein-based light switches used in nature to trigger and regulate various processes. They allow light signals to be converted into metabolic signaling cascades. Various LOV-domain proteins have been characterized in the last few decades and have been used to develop light-sensitive tools in cell biology research. LOV-based applications exploit the light-driven regulation of effector elements to activate signaling pathways, activate genes, or locate proteins within cells. A relatively new application of an engineered small LOV-domain protein called miniSOG (mini singlet oxygen generator) is based on the light-induced formation of reactive oxygen species (ROS). The first miniSOG was engineered from a LOV domain from Arabidopsis thaliana. This engineered 14 kDa light-responsive flavin-containing protein can be exploited as protein tag for the light-triggered localized production of ROS. Such tunable ROS production by miniSOG or similarly redesigned LOV-domains can be of use in studies focused on subcellular phenomena but may also allow new light-fueled catalytic processes. This review provides an overview of the discovery of LOV domains and their development into tools for cell biology. It also highlights recent advancements in engineering LOV domains for various biotechnological applications and cell biology studies.

Abstract: Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, positioning organelles, and modulating cell signaling. Unlike actin filaments and microtubules, IFs have slower dynamics, and current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Perinuclear clustering of vimentin had no strong effect on the actin or microtubule organization, cell spreading, and focal adhesions, but reduced cell stiffness. Mitochondria and endoplasmic reticulum sheets were repositioned together with vimentin, whereas lysosomes were only briefly repositioned and rapidly regained their normal distribution. Keratin was displaced along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.

Abstract: Optogenetic tools have been used in a wide range of microbial engineering applications that benefit from the tunable, spatiotemporal control that light affords. However, the majority of current optogenetic constructs for bacteria respond to blue light, limiting the potential for multichromatic control. In addition, other wavelengths offer potential benefits over blue light, including improved penetration of dense cultures and reduced potential for toxicity. In this study, we introduce OptoCre-REDMAP, a red light inducible Cre recombinase system in Escherichia coli. This system harnesses the plant photoreceptors PhyA and FHY1 and a split version of Cre recombinase to achieve precise control over gene expression and DNA excision. We optimized the design by modifying the start codon of Cre and characterized the impact of different levels of induction to find conditions that produced minimal basal expression in the dark and induced full activation within 4 h of red light exposure. We characterized the system's sensitivity to ambient light, red light intensity, and exposure time, finding OptoCre-REDMAP to be reliable and flexible across a range of conditions. In coculture experiments with OptoCre-REDMAP and the blue light responsive OptoCre-VVD, we found that the systems responded orthogonally to red and blue light inputs. Direct comparisons between red and blue light induction with OptoCre-REDMAP and OptoCre-VVD demonstrated the superior penetration properties of red light. OptoCre-REDMAP's robust and selective response to red light makes it suitable for advanced synthetic biology applications, particularly those requiring precise multichromatic control.

Abstract: Mutations of the cyclin-dependent kinase-like 5 (CDKL5) gene, which encodes a serine/threonine protein kinase, can cause the CDKL5 deficiency disorder (CDD), a severe neurodevelopmental disease characterized by epileptic encephalopathy and neurocognitive impairment. The CDKL5 kinase consists of a catalytic N-terminal domain (NTD) and a less characterized C-terminal domain (CTD). Numerous disease-related mutations truncate CDKL5, leaving the NTD intact while variably shortening the CTD, which highlights the importance of the CTD for CDKL5 function. By systematically analyzing CDKL5 compositional features and evolutionary dynamics, we found that the CTD is a low-complexity region (LCR) highly enriched in serine residues and with a high propensity to undergo liquid-liquid phase separation (LLPS), a biophysical process of condensation controlling protein localization and function. Using a combination of super-resolution imaging, electron microscopy, and molecular and cellular approaches, including optogenetic LLPS induction, we discovered that CDKL5 undergoes LLPS, predominantly driven by its CTD, forming membraneless condensates in neuronal and non-neuronal cells. A CTD internal fragment (CTIF) plays a pivotal LLPS-promoting role, along with the distal portion of the protein. Indeed, two disease-related truncating mutations (S726X and R781X), eliding variable portions of the CTIF, significantly impair LLPS. This impairment is paralleled at the functional level by a reduction in the CDKL5-dependent phosphorylation of EB2, a known CDKL5 target. These findings demonstrate that CDKL5 undergoes LLPS, driven by a CTD region elided by most disease-related truncating mutations. Its loss––through the impairment of CDKL5 LLPS and functional activity––may play a key role in the molecular pathogenesis of CDD.

Abstract: Gene delivery is fundamentally crucial for the genetic manipulation of stem cells. Here, we present a straightforward approach to create a library of charge-neutralized polyethylenimine (PEI)-lipid nanoparticles designed for stem cell transfection. These lipid nanoparticles were formulated using small, branched PEI and lipidic anhydrides. Remarkably, over 15% of the lipid nanoparticles demonstrated high transfection efficiency across various cell types, surpassing the efficiency of both Lipofectamine 2000 and FuGENE HD. A structure-activity analysis indicated that the length and ratio of hydrophobic alkyl substitutions were critical parameters for efficient gene delivery. Notably, the transfection efficiency was higher than that of the original cation PEI. Our optimized PEI-lipid system enabled highly effective plasmid DNA delivery and successfully co-transferred two plasmid DNAs into difficult-to-transfect human embryonic stem cells (hESCs), facilitating optogenetic manipulation within these cells.

Abstract: Collective cell migration is key during development, wound healing, and metastasis and relies on coordinated cell behaviors at the group level. Src kinase is a key signalling protein for the physiological functions of epithelia, as it regulates many cellular processes, including adhesion, motility, and mechanotransduction. Its overactivation is associated with cancer aggressiveness. Here, we take advantage of optogenetics to precisely control Src activation in time and show that its pathological-like activation slows the collective rotation of epithelial cells confined into circular adhesive patches. We interpret velocity, force, and stress data during period of non-activation and period of activation of Src thanks to a hydrodynamic description of the cell assembly as a polar active fluid. Src activation leads to a 2-fold decrease in the ratio of polar angle to friction, which could result from increased adhesiveness at the cell-substrate interface. Measuring internal stress allows us to show that active stresses are subdominant compared to traction forces. Our work reveals the importance of fine-tuning the level of Src activity for coordinated collective behaviors.

Abstract: The post-translational modification of intracellular proteins through O-linked β-N-acetylglucosamine (O-GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O-GlcNAc transferase (OGT), this dynamic modification has an essential role in signal transduction, gene expression, organelle function and systemic physiology. Here, we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression, which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to localize to specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate the downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool for defining the role of O-GlcNAcylation in cell signaling and physiology.

Abstract: In metabolic engineering, increasing chemical production usually involves manipulating the expression levels of key enzymes. However, limited synthetic tools exist for modulating enzyme activity beyond the transcription level. Inspired by natural post-translational mechanisms, we present targeted enzyme degradation mediated by optically controlled nanobodies. We applied this method to a branched biosynthetic pathway, deoxyviolacein, and observed enhanced product specificity and yield. We then extend the biosynthesis pathway to violacein and show how simultaneous degradation of two target enzymes can further shift production profiles. Through the redirection of metabolic flux, we demonstrate how targeted enzyme degradation can be used to minimize unwanted intermediates and boost the formation of desired products.

Abstract: Hsp70 prevents protein aggregation and is cytoprotective, but sustained Hsp70 overexpression is problematic. Therefore, we characterized small molecule agonists that augment Hsp70 activity. Because cumbersome assays were required to assay agonists, we developed cell-based and in vivo assays in which disease-associated consequences of Hsp70 activation can be quantified. One assay uses an optogenetic system in which the formation of TDP-43 inclusions can be controlled, and the second assay employs a zebrafish model for acute kidney injury (AKI). These complementary assays will facilitate future work to identify new Hsp70 agonists as well as optimized agonist derivatives.

Abstract: Light is a powerful and flexible input into engineered biological systems and is particularly well-suited for spatially controlling genetic circuits. While many light-responsive molecular effectors have been developed, there remains a gap in the feasibility of using them to spatially define cell fate. We addressed this problem by employing recombinase as a sensitive light-switchable circuit element which can permanently program cell fate in response to transient illumination. We show that by combining recombinase switches with hardware for precise spatial illumination, large scale heterogeneous populations of cells can be generated in situ with high resolution. We envision that this approach will enable new types of multicellular synthetic circuit engineering where the role of initial cell patterning can be directly studied with both high throughput and tight control.

Abstract: Dysfunction of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) contributes to neurodegeneration, the primary cause of permanent disability in multiple sclerosis (MS). To better understand the role of hnRNP A1 dysfunction in the pathogenesis of neurodegeneration, we utilized optogenetics-driven hnRNP A1 clustering to model its dysfunction in neuron-like differentiated Neuro-2A cells. hnRNP A1 clustering activates the integrated stress response (ISR) and results in a neurodegenerative phenotype marked by decreased neuronal protein translation and neurite loss. Small molecule inhibition of the ISR with either PERKi (GSK2606414) or ISRIB (integrated stress response inhibitor) attenuated both the decrease in neuronal translation and neurite loss, without affecting hnRNP A1 clustering. We then confirmed a strong association between hnRNP A1 clustering and ISR activation in neurons from MS brains. These data illustrate that hnRNP A1 dysfunction promotes neurodegeneration by activation of the ISR in vitro and in vivo, thus revealing a novel therapeutic target to reduce neurodegeneration and subsequent disability in MS.

Abstract: Biomolecular condensates (BMCs), play significant roles in organizing cellular functions in the absence of membranes through phase separation events involving RNA, proteins, and RNA-protein complexes. These membrane-less organelles form dynamic multivalent weak interactions, often involving intrinsically disordered proteins or regions (IDPs/IDRs). However, the nature of these crucial interactions, how most of these organelles are organized and are functional, remains unknown. Aberrant condensates have been implicated in neurodegenerative diseases and various cancers, presenting novel therapeutic opportunities for small molecule condensate modulators. Recent advancements in optogenetic technologies, particularly Corelet, enable precise manipulation of BMC dynamics within living cells, facilitating high-throughput screening for small molecules that target these complex structures. By elucidating the molecular mechanisms governing BMC formation and function, this innovative approach holds promise to unlock therapeutic strategies against previously "undruggable" protein targets, paving the way for effective interventions in disease.

Abstract: Overexpression of a single enzyme in a multigene heterologous pathway may be out of balance with the other enzymes in the pathway, leading to accumulated toxic intermediates, imbalanced carbon flux, reduced productivity of the pathway, or an inhibited growth phenotype. Therefore, optimal, balanced, and synchronized expression levels of enzymes in a particular metabolic pathway is critical to maximize production of desired compounds while maintaining cell fitness in a growing culture. Furthermore, the optimal intracellular concentration of an enzyme is determined by the expression strength, specific timing/duration, and degradation rate of the enzyme. Here, we modulated the intracellular concentration of a key enzyme, namely HMG-CoA synthase (HMGS), in the heterologous mevalonate pathway by tuning its expression level and period of transcription to enhance limonene production in Escherichia coli. Facilitated by the tuned blue-light inducible BLADE/pBad system, we observed that limonene production was highest (160 mg/L) with an intermediate transcription level of HMGS from moderate light illumination (41 au, 150 s ON/150 s OFF) throughout the growth. Owing to the easy penetration and removal of blue-light illumination from the growing culture which is hard to obtain using conventional chemical-based induction, we further explored different induction patterns of HMGS under strong light illumination (2047 au, 300 s ON) for different durations along the growth phases. We identified a specific timing of HMGS expression in the log phase (3-9 h) that led to optimal limonene production (200 mg/L). This is further supported by a mathematical model that predicts several periods of blue-light illumination (3-9 h, 0-9 h, 3-12 h, 0-12 h) to achieve an optimal expression level of HMGS that maximizes limonene production and maintains cell fitness. Compared to moderate and prolonged transcription (41 au, 150 s ON/150 s OFF, 0-73 h), strong but time-limited transcription (2047 au, 300 s ON, 3-9 h) of HMGS could maintain its optimal intracellular concentration and further increased limonene production up to 92% (250 mg/L) in the longer incubation (up to 73 h) without impacting cell fitness. This work has provided new insight into the "right amount" and "just-in-time" expression of a critical metabolite enzyme in the upper module of the mevalonate pathway using optogenetics. This study would complement previous findings in modulating HMGS expression and potentially be applicable to heterologous production of other terpenoids in E. coli.

Abstract: Tight coordination of cell-cell signaling in space and time is vital for self-organization in tissue patterning. During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report the reconstitution of synchronized oscillation in PSM organoids by synthetic cell-cell signaling with designed ligand-receptor pairs. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; non-oscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock, and provides direct evidence of oscillatory cell-cell communication in the segmentation clock.

Abstract: Alternative splicing is a fundamental process that contributes to the functional diversity and complexity of proteins. The regulation of each alternative splicing event involves the coordinated action of multiple RNA-binding proteins, creating a diverse array of alternatively spliced products. Dysregulation of alternative splicing is associated with various diseases, including neurodegeneration. Here we demonstrate that CELF2, a splicing regulator and a GWAS-identified risk factor for Alzheimer’s disease, binds to mRNAs associated with neurodegenerative diseases, with a specific interaction observed in the intron adjacent to exon 10 on Tau mRNA. Loss of CELF2 in the mouse brain results in a decreased inclusion of Tau exon 10, leading to a reduced 4R:3R ratio. Further exploration shows that the hinge domain of CELF2 possesses an intrinsically disordered region (IDR), which mediates CELF2 condensation and function. The functionality of IDR in regulating CELF2 function is underscored by its substitutability with IDRs from FUS and TAF15. Using TurboID we identified proteins that interact with CELF2 through its IDR. We revealed that CELF2 co-condensate with NOVA2 and SFPQ, which coordinate with CELF2 to regulate the alternative splicing of Tau exon 10. A negatively charged residue within the IDR (D388), which is conserved among CELF proteins, is critical for CELF2 condensate formation, interactions with NOVA2 and SFPQ, and function in regulating tau exon 10 splicing. Our data allow us to propose that CELF2 regulates Tau alternative splicing by forming condensates through its IDR with other splicing factors, and that the composition of the proteins within the condensates determines the outcomes of alternative splicing events.

Abstract: Cells use signalling pathways as windows into the environment to gather information, transduce it into their interior, and use it to drive behaviours. MAPK (ERK) is a highly conserved signalling pathway in eukaryotes, directing multiple fundamental cellular behaviours such as proliferation, migration, and differentiation, making it of few central hubs in the signalling circuitry of cells. Despite this versatility of behaviors, population-level measurements have reported low information content (< 1 bit) relayed through the ERK pathway, rendering the population barely able to distinguish the presence or absence of stimuli. Here, we contrast the information transmitted by a single cell and a population of cells. Using a combination of optogenetic experiments, data analysis based on information theory framework, and numerical simulations we quantify the amount of information transduced from the receptor to ERK, from responses to singular, brief and sparse input pulses. We show that single cells are indeed able to resolve between graded stimuli, yielding over 2 bit of information, however showing a large population heterogeneity

Abstract: Spatial organization of microbes in biofilms enables crucial community function such as division of labor. However, quantitative understanding of such emergent community properties remains limited due to a scarcity of tools for patterning heterogeneous biofilms. Here we develop a synthetic optogenetic toolkit 'Multipattern Biofilm Lithography' for rational engineering and orthogonal patterning of multi-strain biofilms, inspired by successive adhesion and phenotypic differentiation in natural biofilms. We apply this toolkit to profile the growth dynamics of heterogeneous biofilm communities, and observe the emergence of spatially modulated commensal relationships due to shared antibiotic protection against the beta-lactam ampicillin. Supported by biophysical modeling, these results yield in-vivo measurements of key parameters, e.g., molecular beta-lactamase production per cell and length scale of antibiotic zone of protection. Our toolbox and associated findings provide quantitative insights into the spatial organization and distributed antibiotic protection within biofilms, with direct implications for future biofilm research and engineering.

Abstract: Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments.

Abstract: Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive, particularly in the endogenous context of developing animals. Here, we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 min) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Abstract: Biomolecular condensates appear throughout cell physiology and pathology, but the specific role of condensation or its dynamics is often difficult to determine. Optogenetics offers an expanding toolset to address these challenges, providing tools to directly control condensation of arbitrary proteins with precision over their formation, dissolution, and patterning in space and time. In this review, we describe the current state of the field for optogenetic control of condensation. We survey the proteins and their derivatives that form the foundation of this toolset, and we discuss the factors that distinguish them to enable appropriate selection for a given application. We also describe recent examples of the ways in which optogenetic condensation has been used in both basic and applied studies. Finally, we discuss important design considerations when engineering new proteins for optogenetic condensation, and we preview future innovations that will further empower this toolset in the coming years.

Abstract: Chimeric antigen receptor T cell therapies have achieved great success in eradicating some liquid tumors, whereas the preclinical results in treating solid tumors have proven less decisive. One of the principal challenges in solid tumor treatment is the physical barrier composed of a dense extracellular matrix, which prevents immune cells from penetrating the tissue to attack intratumoral cancer cells. Here, we improve immune cell infiltration into solid tumors by manipulating septin-7 functions in cells. Using protein allosteric design, we reprogram the three-dimensional structure of septin-7 and insert a blue light-responsive light-oxygen-voltage-sensing domain 2 (LOV2), creating a light-controllable septin-7-LOV2 hybrid protein. Blue light inhibits septin-7 function in live cells, inducing extended cell protrusions and cell polarization, enhancing cell transmigration efficiency through confining spaces. We genetically edited human natural killer cell line (NK92) and mouse primary CD8+ T-cells expressing the engineered protein, and we demonstrated improved penetration and cytotoxicity against various tumor spheroid models. Our proposed strategy to enhance immune cell infiltration is compatible with other methodologies and therefore, could be used in combination to further improve cell-based immunotherapies against solid tumors.