Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 25 of 332 results
1.

Optogenetic Regulation of EphA1 RTK Activation and Signaling.

blue CRY2olig HEK293T Neuro-2a Signaling cascade control Control of cytoskeleton / cell motility / cell shape
bioRxiv, 20 Feb 2024 DOI: 10.1101/2024.02.06.579139 Link to full text
Abstract: Eph receptors are ubiquitous class of transmembrane receptors that mediate cell-cell communication, proliferation, differentiation, and migration. EphA1 receptors specifically play an important role in angiogenesis, fetal development, and cancer progression; however, studies of this receptor can be challenging as its ligand, ephrinA1, binds and activates several EphA receptors simultaneously. Optogenetic strategies could be applied to circumvent this requirement for ligand activation and enable selective activation of the EphA1 subtype. In this work, we designed and tested several iterations of an optogenetic EphA1 - Cryptochrome 2 (Cry2) fusion, investigating their capacity to mimic EphA1-dependent signaling in response to light activation. We then characterized the key cell signaling target of MAPK phosphorylation activated in response to light stimulation. The optogenetic regulation of Eph receptor RTK signaling without the need for external stimulus promises to be an effective means of controlling individual Eph receptor-mediated activities and creates a path forward for the identification of new Eph-dependent functions.
2.

OptoREACT: Optogenetic Receptor Activation on Nonengineered Human T Cells.

red PhyB/PIF6 HEK293T human T cells Jurkat Signaling cascade control Extracellular optogenetics
ACS Synth Biol, 9 Feb 2024 DOI: 10.1021/acssynbio.3c00518 Link to full text
Abstract: Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.
3.

Using split protein reassembly strategy to optically control PLD enzymatic activity.

blue CRY2/CIB1 iLID HEK293T HeLa Signaling cascade control Organelle manipulation
bioRxiv, 30 Jan 2024 DOI: 10.1101/2024.01.27.577557 Link to full text
Abstract: Phospholipase D (PLD) and phosphatidic acid (PA) play a spatio-temporal role in regulating diverse cellular activities. Although current methodologies enable optical control of the subcellular localization of PLD and by which influence local PLD enzyme activity, the overexpression of PLD elevates the basal PLD enzyme activity and further leads to increased PA levels in cells. In this study, we employed a split protein reassembly strategy and optogenetic techniques to modify superPLD (a PLDPMF variant with a high basal activity). We splited this variants into two HKD domains and fused these domains with optogenetic elements and by which we achieved light-mediated dimerization of the two HKD proteins and then restored the PLD enzymatic activity.
4.

Programmable RNA base editing with photoactivatable CRISPR-Cas13.

blue Magnets HEK293T HeLa HT-1080 MCF7 mouse in vivo Neuro-2a Nucleic acid editing
Nat Commun, 22 Jan 2024 DOI: 10.1038/s41467-024-44867-2 Link to full text
Abstract: CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development.
5.

Rapid Optogenetic Clustering in the Cytoplasm with BcLOVclust.

blue BcLOV4 CRY2/CRY2 HEK293T Signaling cascade control
J Mol Biol, 20 Jan 2024 DOI: 10.1016/j.jmb.2024.168452 Link to full text
Abstract: Protein clustering is a powerful form of optogenetic control, yet remarkably few proteins are known to oligomerize with light. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple BcLOV4 clustering to membrane binding, allowing us to engineer a variant that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant-called BcLOVclust-clustered over many cycles with substantially faster clustering and de-clustering kinetics compared to the widely used optogenetic clustering protein Cry2. The magnitude of clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by selecting the appropriate fluorescent protein to which it was fused. Like wt BcLOV4, BcLOVclust activity was sensitive to temperature: light-induced clusters spontaneously dissolved at a rate that increased with temperature despite constant illumination. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. While its usage is currently best suited in cells and organisms that can be cultured below ∼30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.
6.

Development of an optogenetics tool, Opto-RANK, for control of osteoclast differentiation using blue light.

blue CRY2/CIB1 CRY2clust HEK293T RAW264.7 Signaling cascade control
Sci Rep, 19 Jan 2024 DOI: 10.1038/s41598-024-52056-w Link to full text
Abstract: Optogenetics enables precise regulation of intracellular signaling in target cells. However, the application of optogenetics to induce the differentiation of precursor cells and generate mature cells with specific functions has not yet been fully explored. Here, we focused on osteoclasts, which play an important role in bone remodeling, to develop a novel optogenetics tool, Opto-RANK, which can manipulate intracellular signals involved in osteoclast differentiation and maturation using blue light. We engineered Opto-RANK variants, Opto-RANKc and Opto-RANKm, and generated stable cell lines through retroviral transduction. Differentiation was induced by blue light, and various assays were conducted for functional analysis. Osteoclast precursor cells expressing Opto-RANK differentiated into multinucleated giant cells on light exposure and displayed upregulation of genes normally induced in differentiated osteoclasts. Furthermore, the differentiated cells exhibited bone-resorbing activities, with the possibility of spatial control of the resorption by targeted light illumination. These results suggested that Opto-RANK cells differentiated by light possess the features of osteoclasts, both morphological and functional. Thus, Opto-RANK should be useful for detailed spatiotemporal analysis of intracellular signaling during osteoclast differentiation and the development of new therapies for various bone diseases.
7.

Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag.

blue iLID HEK293T NIH/3T3 Organelle manipulation
bioRxiv, 17 Jan 2024 DOI: 10.1101/2024.01.16.575860 Link to full text
Abstract: Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function—dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles, a major question in cell biology and drug development. Here we report an optogenetic approach to selectively dissolve a condensate of interest in a reversible and spatially controlled manner. We show that light-gated recruitment of maltose-binding protein (MBP), a commonly used solubilizing domain in protein purification, results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP, showing that disrupting condensation of the oncogenic fusion protein FUS-CHOP results in reversion of FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.
8.

An RNA Motif That Enables Optozyme Control and Light-Dependent Gene Expression in Bacteria and Mammalian Cells.

blue PAL E. coli HEK293T Transgene expression
Adv Sci (Weinh), 16 Jan 2024 DOI: 10.1002/advs.202304519 Link to full text
Abstract: The regulation of gene expression by light enables the versatile, spatiotemporal manipulation of biological function in bacterial and mammalian cells. Optoribogenetics extends this principle by molecular RNA devices acting on the RNA level whose functions are controlled by the photoinduced interaction of a light-oxygen-voltage photoreceptor with cognate RNA aptamers. Here light-responsive ribozymes, denoted optozymes, which undergo light-dependent self-cleavage and thereby control gene expression are described. This approach transcends existing aptamer-ribozyme chimera strategies that predominantly rely on aptamers binding to small molecules. The optozyme method thus stands to enable the graded, non-invasive, and spatiotemporally resolved control of gene expression. Optozymes are found efficient in bacteria and mammalian cells and usher in hitherto inaccessible optoribogenetic modalities with broad applicability in synthetic and systems biology.
9.

Bidirectional Allosteric Coupling between PIP2 Binding and the Pore of the Oncochannel TRPV6.

blue CRY2/CIB1 HEK293 Immediate control of second messengers
Int J Mol Sci, 3 Jan 2024 DOI: 10.3390/ijms25010618 Link to full text
Abstract: The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.
10.

Optogenetic induction of caspase-8 mediated apoptosis by employing Arabidopsis cryptochrome 2.

blue CRY2/CIB1 CRY2/CRY2 HEK293T HeLa Signaling cascade control Cell death
Sci Rep, 27 Dec 2023 DOI: 10.1038/s41598-023-50561-y Link to full text
Abstract: Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.
11.

Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid interference system.

blue AsLOV2 E. coli HEK293T Nucleic acid editing
Nucleic Acids Res, 11 Dec 2023 DOI: 10.1093/nar/gkad995 Link to full text
Abstract: Anti-CRISPR (Acr) proteins are encoded by mobile genetic elements to overcome the CRISPR immunity of prokaryotes, displaying promises as controllable tools for modulating CRISPR-based applications. However, characterizing novel anti-CRISPR proteins and exploiting Acr-related technologies is a rather long and tedious process. Here, we established a versatile plasmid interference with CRISPR interference (PICI) system in Escherichia coli for rapidly characterizing Acrs and developing Acr-based technologies. Utilizing the PICI system, we discovered two novel type II-A Acrs (AcrIIA33 and AcrIIA34), which can inhibit the activity of SpyCas9 by affecting DNA recognition of Cas9. We further constructed a circularly permuted AcrIIA4 (cpA4) protein and developed optogenetically engineered, robust AcrIIA4 (OPERA4) variants by combining cpA4 with the light-oxygen-voltage 2 (LOV2) blue light sensory domain. OPERA4 variants are robust light-dependent tools for controlling the activity of SpyCas9 by approximately 1000-fold change under switching dark-light conditions in prokaryotes. OPERA4 variants can achieve potent light-controllable genome editing in human cells as well. Together, our work provides a versatile screening system for characterizing Acrs and developing the Acr-based controllable tools.
12.

Design and Engineering of Light-Induced Base Editors Facilitating Genome Editing with Enhanced Fidelity.

blue Magnets E. coli HEK293T Nucleic acid editing
Adv Sci (Weinh), 1 Dec 2023 DOI: 10.1002/advs.202305311 Link to full text
Abstract: Base editors, which enable targeted locus nucleotide conversion in genomic DNA without double-stranded breaks, have been engineered as powerful tools for biotechnological and clinical applications. However, the application of base editors is limited by their off-target effects. Continuously expressed deaminases used for gene editing may lead to unwanted base alterations at unpredictable genomic locations. In the present study, blue-light-activated base editors (BLBEs) are engineered based on the distinct photoswitches magnets that can switch from a monomer to dimerization state in response to blue light. By fusing the N- and C-termini of split DNA deaminases with photoswitches Magnets, efficient A-to-G and C-to-T base editing is achieved in response to blue light in prokaryotic and eukaryotic cells. Furthermore, the results showed that BLBEs can realize precise blue light-induced gene editing across broad genomic loci with low off-target activity at the DNA- and RNA-level. Collectively, these findings suggest that the optogenetic utilization of base editing and optical base editors may provide powerful tools to promote the development of optogenetic genome engineering.
13.

Spatiotemporal control of RNA metabolism and CRISPR-Cas functions using engineered photoswitchable RNA-binding proteins.

blue VVD HEK293 HEK293T mouse in vivo zebrafish in vivo
Nat Protoc, 30 Nov 2023 DOI: 10.1038/s41596-023-00920-w Link to full text
Abstract: RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR-Cas system to optogenetic control. The protocols typically take 2-3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR-Cas functions.
14.

Optogenetic-mediated induction and monitoring of α-synuclein aggregation in cellular models of Parkinson's disease.

blue CRY2olig HEK293T
STAR Protoc, 21 Nov 2023 DOI: 10.1016/j.xpro.2023.102738 Link to full text
Abstract: Studying Parkinson's disease (PD) is complex due to a lack of cellular models mimicking key aspects of protein pathology. Here, we present a protocol for inducing and monitoring α-synuclein aggregation in living cells using optogenetics. We describe steps for plasmid transduction, biochemical validation, immunocytochemistry, and live-cell confocal imaging. These induced aggregates fulfill the cardinal features of authentic protein inclusions observed in PD-diseased brains and offer a tool to study the role of protein aggregation in neurodegeneration. For complete details on the use and execution of this protocol, please refer to Bérard et al.1.
15.

A single-component, light-assisted uncaging switch for endoproteolytic release.

blue violet CRY2/CIB1 iLID PhoCl HEK293T primary rat hippocampal neurons Signaling cascade control Transgene expression
Nat Chem Biol, 16 Nov 2023 DOI: 10.1038/s41589-023-01480-6 Link to full text
Abstract: Proteases function as pivotal molecular switches, initiating numerous biological events. Notably, potyviral protease, derived from plant viruses, has emerged as a trusted proteolytic switch in synthetic biological circuits. To harness their capabilities, we have developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), by employing a circularly permutated tobacco etch virus protease and a blue-light-gated substrate, which are connected by fine-tuned intermodular linkers. As a single-component system, LAUNCHER exhibits a superior signal-to-noise ratio compared with multi-component systems, enabling precise and user-controllable release of payloads. This characteristic renders LAUNCHER highly suitable for diverse cellular applications, including transgene expression, tailored subcellular translocation and optochemogenetics. Additionally, the plug-and-play integration of LAUNCHER into existing synthetic circuits facilitates the enhancement of circuit performance. The demonstrated efficacy of LAUNCHER in improving existing circuitry underscores its significant potential for expanding its utilization in various applications.
16.

Light induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

blue EL222 HEK293FT HEK293T mouse in vivo mouse T cells Transgene expression Endogenous gene expression Nucleic acid editing
bioRxiv, 10 Nov 2023 DOI: 10.1101/2023.11.09.566272 Link to full text
Abstract: There is currently a lack of tools capable of perturbing genes in both a precise and spatiotemporal fashion. CRISPR’s ease of use and flexibility, 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, BLU-VIPR, that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of gRNA production. This simplifies spatiotemporal gene perturbation and works in vivo with cells previously intractable to optogenetic gene editing. We engineered BLU-VIPR around a new potent blue-light activated transcription factor and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single mRNA transcript, allowing for optogenetic gene editing in T lymphocytes in vivo.
17.

Near-Infrared Optogenetic Module for Conditional Protein Splicing.

red DrBphP MagRed HEK293T HeLa Transgene expression Cell death
J Mol Biol, 8 Nov 2023 DOI: 10.1016/j.jmb.2023.168360 Link to full text
Abstract: Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue penetration, holds particular promise. However, the optogenetic control of polypeptide bond formation has not yet been developed. In this study, we introduce a NIR optogenetic module for conditional protein splicing (CPS) based on the gp41-1 intein. We optimized the module to minimize background signals in the darkness and to maximize the contrast between light and dark conditions. Next, we engineered a NIR CPS gene expression system based on the protein ligation of a transcription factor. We applied the NIR CPS for light-triggered protein cleavage to activate gasdermin D, a pore-forming protein that induces pyroptotic cell death. Our NIR CPS optogenetic module represents a promising tool for controlling molecular processes through covalent protein linkage and cleavage.
18.

Turn-On Protein Switches for Controlling Actin Binding in Cells.

blue AsLOV2 HEK293T HeLa MDCK Control of cytoskeleton / cell motility / cell shape
bioRxiv, 26 Oct 2023 DOI: 10.1101/2023.10.26.561921 Link to full text
Abstract: Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP’s influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into new structures to control cell and tissue shape and behavior.
19.

A programmable protease-based protein secretion platform for therapeutic applications.

blue red BphS CRY2/CIB1 Magnets PhyA/FHY1 Hana3A HEK293T hMSCs mouse IPSCs Control of vesicular transport
Nat Chem Biol, 23 Oct 2023 DOI: 10.1038/s41589-023-01433-z Link to full text
Abstract: Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.
20.

Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice.

blue CRY2/CIB1 CRY2/CRY2 CRY2olig HEK293T U-2 OS Transgene expression Endogenous gene expression Organelle manipulation
bioRxiv, 16 Oct 2023 DOI: 10.1101/2023.10.16.562453 Link to full text
Abstract: Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, we systematically tune the material properties of optogenetically induced transcription factor condensates and probe their impact on the activation of target promoters. We demonstrate that rather liquid condensates correlate with increased gene expression levels, whereas a gradual transition to more stiff condensates converts otherwise activating transcription factors into dominant negative inhibitors. We demonstrate the general nature of these findings in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. We observe these effects for both transgenic and cell-endogenous promoters. Our findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in (opto-)genetic engineering and synthetic biology.
21.

OptoProfilin: A Single Component Biosensor of Applied Cellular Stress.

blue CRY2/CRY2 HEK293T HeLa Neuro-2a NIH/3T3 Control of cytoskeleton / cell motility / cell shape
bioRxiv, 4 Oct 2023 DOI: 10.1101/2023.10.04.560945 Link to full text
Abstract: The actin cytoskeleton is a biosensor of cellular stress and a potential prognosticator of human disease. In particular, aberrant cytoskeletal structures such as cofilin-actin rods and stress granules formed in response to energetic and oxidative stress are closely linked to neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and ALS. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, neurodegenerative disease progression, remains an open question. In this work, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We demonstrate that this optically activated profilin (‘OptoProfilin’) can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists. While a large body of work closely links profilin activity with cellular stress and neurodegenerative disease, this, to our knowledge, is the first example of profilin as an optogenetic biosensor of stress-induced changes in the cytoskeleton.
22.

Spatiotemporal, optogenetic control of gene expression in organoids.

blue CRY2/CIB1 Magnets HEK293T human IPSCs Endogenous gene expression Nucleic acid editing
Nat Methods, 21 Sep 2023 DOI: 10.1038/s41592-023-01986-w Link to full text
Abstract: Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.
23.

An optogenetic method for the controlled release of single molecules.

violet PhoCl CHO-K1 CV-1 EL4 HEK293T Signaling cascade control Organelle manipulation
bioRxiv, 17 Sep 2023 DOI: 10.1101/2023.09.16.557871 Link to full text
Abstract: We developed a system for optogenetic release of single molecules in live cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for the controlled delivery of functional proteins to cytosol and plasma membrane in amounts compatible with single molecule imaging, greatly simplifying access to single molecule microscopy of any protein in live cells. Furthermore, we could reconstitute cellular functions such as ion conductance by delivering BK and VRAC ion channels to the plasma membrane. Finally, we could induce NF-kB signaling in T-Lymphoblasts stimulated by IL-1 by controlled release of a signaling protein that had been knocked-out in the same cells. We observed light induced formation of functional inflammatory signaling complexes that could trigger IKK phosphorylation in single cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single molecule level.
24.

Photoactivatable base editors for spatiotemporally controlled genome editing in vivo.

blue AsLOV2 CRY2/CIB1 Magnets HEK293T mouse in vivo Transgene expression Nucleic acid editing
Biomaterials, 13 Sep 2023 DOI: 10.1016/j.biomaterials.2023.122328 Link to full text
Abstract: CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.
25.

Diya – a universal light illumination platform for multiwell plate cultures.

blue green CcaS/CcaR CRY2/CIB1 EL222 Magnets VVD E. coli HEK293T HeLa S. cerevisiae Transgene expression
iScience, 9 Sep 2023 DOI: 10.1016/j.isci.2023.107862 Link to full text
Abstract: Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform – Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially-designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.
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