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.

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A biological camera that captures and stores images directly into DNA.

blue red PhyB/PIF3 VVD E. coli Nucleic acid editing Multichromatic
Nat Commun, 3 Jul 2023 DOI: 10.1038/s41467-023-38876-w Link to full text
Abstract: The increasing integration between biological and digital interfaces has led to heightened interest in utilizing biological materials to store digital data, with the most promising one involving the storage of data within defined sequences of DNA that are created by de novo DNA synthesis. However, there is a lack of methods that can obviate the need for de novo DNA synthesis, which tends to be costly and inefficient. Here, in this work, we detail a method of capturing 2-dimensional light patterns into DNA, by utilizing optogenetic circuits to record light exposure into DNA, encoding spatial locations with barcoding, and retrieving stored images via high-throughput next-generation sequencing. We demonstrate the encoding of multiple images into DNA, totaling 1152 bits, selective image retrieval, as well as robustness to drying, heat and UV. We also demonstrate successful multiplexing using multiple wavelengths of light, capturing 2 different images simultaneously using red and blue light. This work thus establishes a 'living digital camera', paving the way towards integrating biological systems with digital devices.

Multidimensional characterization of inducible promoters and a highly light-sensitive LOV-transcription factor.

blue red EL222 PhyB/PIF3 S. cerevisiae Transgene expression
Nat Commun, 27 Jun 2023 DOI: 10.1038/s41467-023-38959-8 Link to full text
Abstract: The ability to independently control the expression of different genes is important for quantitative biology. Using budding yeast, we characterize GAL1pr, GALL, MET3pr, CUP1pr, PHO5pr, tetOpr, terminator-tetOpr, Z3EV, blue-light inducible optogenetic systems El222-LIP, El222-GLIP, and red-light inducible PhyB-PIF3. We report kinetic parameters, noise scaling, impact on growth, and the fundamental leakiness of each system using an intuitive unit, maxGAL1. We uncover disadvantages of widely used tools, e.g., nonmonotonic activity of MET3pr and GALL, slow off kinetics of the doxycycline- and estradiol-inducible systems tetOpr and Z3EV, and high variability of PHO5pr and red-light activated PhyB-PIF3 system. We introduce two previously uncharacterized systems: strongLOV, a more light-sensitive El222 mutant, and ARG3pr, which is induced in the absence of arginine or presence of methionine. To demonstrate fine control over gene circuits, we experimentally tune the time between cell cycle Start and mitosis, artificially simulating near-wild-type timing. All strains, constructs, code, and data ( ) are made available.

A photoreceptor-based hydrogel with red light-responsive reversible sol-gel transition as transient cellular matrix.

red PhyB/PIF6 in vitro Extracellular optogenetics
bioRxiv, 23 May 2023 DOI: 10.1101/2023.04.04.535523 Link to full text
Abstract: Hydrogels with adjustable mechanical properties have been engineered as matrices for mammalian cells and allow the dynamic, mechano-responsive manipulation of cell fate and function. Recent research yielded hydrogels, where biological photoreceptors translated optical signals into a reversible and adjustable change in hydrogel mechanics. While their initial application provided important insights into mechanobiology, broader implementation is limited by a small dynamic range of addressable stiffness. Here, we overcome this limitation by developing a photoreceptor-based hydrogel with reversibly adjustable stiffness from 800 Pa to the sol state. The hydrogel is based on star-shaped polyethylene glycol, functionalized with the red/far-red light photoreceptor phytochrome B (PhyB), or phytochrome-interacting factor 6 (PIF6). Upon illumination with red light, PhyB heterodimerizes with PIF6, thus crosslinking the polymers and resulting in gelation. However, upon illumination with far-red light, the proteins dissociate and trigger a complete gel-to-sol transition. We comprehensively characterize the hydrogel’s light-responsive mechanical properties and apply it as reversible extracellular matrix for the spatiotemporally controlled deposition of mammalian cells within a microfluidic chip. We anticipate that this technology will open new avenues for the site- and time-specific positioning of cells and will contribute to overcome spatial restrictions.

PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae.

red PhyB/PIF3 S. cerevisiae Endogenous gene expression
ACS Synth Biol, 4 Apr 2023 DOI: 10.1021/acssynbio.2c00517 Link to full text
Abstract: Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, e.g., in order to rewire metabolic fluxes. Here, we introduce the programmable red light switch PhiReX 2.0, which can rewire metabolic fluxes by targeting endogenous promoter sequences through single-guide RNAs (sgRNAs) and activate gene expression in Saccharomyces cerevisiae upon red light stimulation. The split transcription factor is built from the plant-derived optical dimer PhyB and PIF3, which is fused to a DNA-binding domain based on the catalytically dead Cas9 protein (dCas9) and a transactivation domain. This design combines at least two major advantages: first, the sgRNAs, guiding dCas9 to the promoter of interest, can be exchanged in an efficient and straightforward Golden Gate-based cloning approach, which allows for rational or randomized combination of up to four sgRNAs in a single expression array. Second, target gene expression can be rapidly upregulated by short red light pulses in a light dose-dependent manner and returned to the native expression level by applying far-red light without interfering with the cell culture. Using the native yeast gene CYC1 as an example, we demonstrated that PhiReX 2.0 can upregulate CYC1 gene expression by up to 6-fold in a light intensity-dependent and reversible manner using a single sgRNA.

Optical Control of Cell Signaling with Red/Far-Red Light-Responsive Optogenetic Tools in Caenorhabditis elegans.

red PhyB/PIF3 C. elegans in vivo Immediate control of second messengers
ACS Synth Biol, 20 Feb 2023 DOI: 10.1021/acssynbio.2c00461 Link to full text
Abstract: Optogenetic techniques have been intensively applied to the nematode Caenorhabditis elegans to investigate its neural functions. However, as most of these optogenetics are responsive to blue light and the animal exhibits avoidance behavior to blue light, the application of optogenetic tools responsive to longer wavelength light has been eagerly anticipated. In this study, we report the implementation in C. elegans of a phytochrome-based optogenetic tool that responds to red/near-infrared light and manipulates cell signaling. We first introduced the SynPCB system, which enabled us to synthesize phycocyanobilin (PCB), a chromophore for phytochrome, and confirmed the biosynthesis of PCB in neurons, muscles, and intestinal cells. We further confirmed that the amount of PCBs synthesized by the SynPCB system was sufficient for photoswitching of phytochrome B (PhyB)-phytochrome interacting factor 3 (PIF3). In addition, optogenetic elevation of intracellular Ca2+ levels in intestinal cells induced a defecation motor program. These SynPCB system and phytochrome-based optogenetic techniques would be of great value in elucidating the molecular mechanisms underlying C. elegans behaviors.

Opto-APC: Engineering of cells that display phytochrome B on their surface for optogenetic studies of cell-cell interactions.

red PhyB/PIF6 HEK293T Jurkat Raji Control of cell-cell / cell-material interactions Extracellular optogenetics
Front Mol Biosci, 20 Feb 2023 DOI: 10.3389/fmolb.2023.1143274 Link to full text
Abstract: The kinetics of a ligand-receptor interaction determine the responses of the receptor-expressing cell. One approach to experimentally and reversibly change this kinetics on demand is optogenetics. We have previously developed a system in which the interaction of a modified receptor with an engineered ligand can be controlled by light. In this system the ligand is a soluble Phytochrome B (PhyB) tetramer and the receptor is fused to a mutated PhyB-interacting factor (PIFS). However, often the natural ligand is not soluble, but expressed as a membrane protein on another cell. This allows ligand-receptor interactions in two dimensions. Here, we developed a strategy to generate cells that display PhyB as a membrane-bound protein by expressing the SpyCatcher fused to a transmembrane domain in HEK-293T cells and covalently coupling purified PhyB-SpyTag to these cells. As proof-of-principle, we use Jurkat T cells that express a GFP-PIFS-T cell receptor and show that these cells can be stimulated by the PhyB-coupled HEK-293T cells in a light dependent manner. Thus, we call the PhyB-coupled cells opto-antigen presenting cells (opto-APCs). Our work expands the toolbox of optogenetic technologies, allowing two-dimensional ligand-receptor interactions to be controlled by light.

Orthogonal Light-Dependent Membrane Adhesion Induces Social Self-Sorting and Member-Specific DNA Communication in Synthetic Cell Communities.

blue red iLID PhyB/PIF6 in vitro Extracellular optogenetics Multichromatic
Small, 4 Jan 2023 DOI: 10.1002/smll.202206474 Link to full text
Abstract: Developing orthogonal chemical communication pathways in diverse synthetic cell communities is a considerable challenge due to the increased crosstalk and interference associated with large numbers of different types of sender-receiver pairs. Herein, the authors control which sender-receiver pairs communicate in a three-membered community of synthetic cells through red and blue light illumination. Semipermeable protein-polymer-based synthetic cells (proteinosomes) with complementary membrane-attached protein adhesion communicate through single-stranded DNA oligomers and synergistically process biochemical information within a community consisting of one sender and two different receiver populations. Different pairs of red and blue light-responsive protein-protein interactions act as membrane adhesion mediators between the sender and receivers such that they self-assemble and socially self-sort into different multicellular structures under red and blue light. Consequently, distinct sender-receiver pairs come into the signaling range depending on the light illumination and are able to communicate specifically without activation of the other receiver population. Overall, this work shows how photoswitchable membrane adhesion gives rise to different self-sorting protocell patterns that mediate member-specific DNA-based communication in ternary populations of synthetic cells and provides a step towards the design of orthogonal chemical communication networks in diverse communities of synthetic cells.

Integration of light and temperature sensing by liquid-liquid phase separation of phytochrome B.

blue red CRY2/CRY2 PhyB/PIF3 HEK293T Organelle manipulation
Mol Cell, 12 Jun 2022 DOI: 10.1016/j.molcel.2022.05.026 Link to full text
Abstract: Light and temperature in plants are perceived by a common receptor, phytochrome B (phyB). How phyB distinguishes these signals remains elusive. Here, we report that phyB spontaneously undergoes phase separation to assemble liquid-like droplets. This capacity is driven by its C terminus through self-association, whereas the intrinsically disordered N-terminal extension (NTE) functions as a biophysical modulator of phase separation. Light exposure triggers a conformational change to subsequently alter phyB condensate assembly, while temperature sensation is directly mediated by the NTE to modulate the phase behavior of phyB droplets. Multiple signaling components are selectively incorporated into phyB droplets to form concentrated microreactors, allowing switch-like control of phyB signaling activity through phase transitions. Therefore, light and temperature cues are separately read out by phyB via allosteric changes and spontaneous phase separation, respectively. We provide a conceptual framework showing how the distinct but highly correlated physical signals are interpreted and sorted by one receptor.

Spatially Defined Gene Delivery into Native Cells with the Red Light-Controlled OptoAAV Technology.

red PhyB/PIF6 A-431 in vitro
Curr Protoc, Jun 2022 DOI: 10.1002/cpz1.440 Link to full text
Abstract: The OptoAAV technology allows spatially defined delivery of transgenes into native target cells down to single-cell resolution by the illumination with cell-compatible and tissue-penetrating red light. The system is based on an adeno-associated viral (AAV) vector of serotype 2 with an engineered capsid (OptoAAV) and a photoreceptor-containing adapter protein mediating the interaction of the OptoAAV with the surface of the target cell in response to low doses of red and far-red light. In this article, we first provide detailed protocols for the production, purification, and analysis of the OptoAAV and the adapter protein. Afterward, we describe in detail the application of the OptoAAV system for the light-controlled transduction of human cells with global and patterned illumination. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production, purification, and analysis of PhyB-DARPinEGFR adapter protein Basic Protocol 2: Production, purification, and analysis of OptoAAV Basic Protocol 3: Red light-controlled viral transduction with the OptoAAV system Support Protocol: Spatially resolved transduction of two transgenes with the OptoAAV system.

Engineered Cas9 extracellular vesicles as a novel gene editing tool.

blue red CRY2/CIB1 Magnets PhyB/PIF6 VVD HEK293T Nucleic acid editing
J Extracell Vesicles, May 2022 DOI: 10.1002/jev2.12225 Link to full text
Abstract: Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

Light-inducible T cell engagers trigger, tune and shape the activation of primary T cells.

red PhyB/PIF6 human T cells Extracellular optogenetics
bioRxiv, 15 Apr 2022 DOI: 10.1101/2022.04.15.488452 Link to full text
Abstract: Cells perceive overtime complex sequences of receptor stimulation that they integrate to mount an appropriate response. Yet, the influence of signal dynamics on cell responses has been poorly characterized due to technical limitations. Here, we present a generalizable approach to control receptor stimulation on unmodified primary cells. Indeed, for applications on primary murine T cells, we have engineered the LiTe system, a new recombinant optogenetics-based Light-inducible T cell engager which allows tunable and reversible spatiotemporal control of the T Cell Receptor (TCR) stimulation. We also provided in vitro evidence that this system enables efficient T cell activation with light, leading to cytokine secretion or tumor cell killing. Using specific time-gated stimulations, we have been able to orient the outcome of the activation of T cells. Overall, the LiTe system constitutes a versatile ON/OFF molecular switch allowing to decipher the cellular response to stimulation dynamics. Its original control over T cell activation opens new avenues for future precision cancer immunotherapy.

OptoAssay - Light-controlled Dynamic Bioassay Using Optogenetic Switches.

red PhyB/PIF6 in vitro Extracellular optogenetics
bioRxiv, 8 Nov 2021 DOI: 10.1101/2021.11.06.467572 Link to full text
Abstract: Circumventing the limitations of current bioassays, we introduce the first light-controlled assay, the OptoAssay, towards wash- and pump-free point-of-care diagnostics. Extending the capabilities of standard bioassays with light-dependent and reversible interaction of optogenetic switches, OptoAssays enable a bi-directional movement of assay components, only by changing the wavelength of light. Combined with smartphones, OptoAssays obviate the need for external flow control systems like pumps or valves and signal readout devices.

Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors.

red PhyB/PIF6 A-431 A549 CHO-K1 HEK293T HeLa MDA-MB-231 MDA-MB-453 SK-OV-3 Extracellular optogenetics
Sci Adv, 16 Jun 2021 DOI: 10.1126/sciadv.abf0797 Link to full text
Abstract: Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

Cross-TCR Antagonism Revealed by Optogenetically Tuning the Half-Life of the TCR Ligand Binding.

red PhyB/PIF6 Jurkat Signaling cascade control
Int J Mol Sci, 6 May 2021 DOI: 10.3390/ijms22094920 Link to full text
Abstract: Activation of T cells by agonistic peptide-MHC can be inhibited by antagonistic ones. However, the exact mechanism remains elusive. We used Jurkat cells expressing two different TCRs and tested whether stimulation of the endogenous TCR by agonistic anti-Vβ8 antibodies can be modulated by ligand-binding to the second, optogenetic TCR. The latter TCR uses phytochrome B tetramers (PhyBt) as ligand, the binding half-life of which can be altered by light. We show that this half-life determined whether the PhyBt acted as a second agonist (long half-life), an antagonist (short half-life) or did not have any influence (very short half-life) on calcium influx. A mathematical model of this cross-antagonism shows that a mechanism based on an inhibitory signal generated by early recruitment of a phosphatase and an activating signal by later recruitment of a kinase explains the data.

Photo-dependent membrane-less organelles formed from plant phyB and PIF6 proteins in mammalian cells.

red PhyB/PIF6 HEK293
Int J Biol Macromol, 11 Feb 2021 DOI: 10.1016/j.ijbiomac.2021.02.075 Link to full text
Abstract: Plant photobodies are the membrane-less organelles (MLOs) that can be generated by protein-protein interactions between active form of phytochrome B (phyB) and phytochrome-interacting factors (PIFs). These organelles regulate plant photomorphogenesis. In this study, we developed two chimeric proteins with fluorescent proteins, phyB fused to EGFP and PIF6 fused to mCherry, and investigated their exogenous expression in mammalian cells by confocal fluorescence microscopy. Results showed that irradiation with diffused 630-nm light induced formation and subsequent increase in sizes of the MLOs. The assembly and disassembly of the photo-inducible MLOs in the mammalian cell cytoplasm obeyed the laws inherent in the concentration-dependent phase separation of biopolymers. The sizes of MLOs formed from phyB and PIF6 in mammalian cells corresponded to the sizes of the so-called "early" photobodies in plant cells. These results suggested that the first step for the formation of plant photobodies might be based on the light-dependent liquid-liquid phase separation of PIFs and other proteins that can specifically interact with the active form of phyB. The developed chimeric proteins in principle can be used to control the assembly and disassembly of photo-inducible MLOs, and thereby to regulate various intracellular processes in mammalian cells.

Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice.

blue red CRY2/CIB1 CRY2/CRY2 PhyB/PIF6 HEK293 mouse in vivo U-2 OS Transgene expression
Sci Adv, 1 Jan 2021 DOI: 10.1126/sciadv.abd3568 Link to full text
Abstract: Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse.

blue red CRY2/CIB1 Magnets PhyB/PIF3 VVD HEK293T HeLa HEp-2 mouse in vivo SH-SY5Y Nucleic acid editing
Proc Natl Acad Sci U S A, 14 Dec 2020 DOI: 10.1073/pnas.2003991117 Link to full text
Abstract: Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

Improvement of Phycocyanobilin Synthesis for Genetically Encoded Phytochrome-Based Optogenetics.

red PhyB/PIF3 HeLa mESCs Xenopus in vivo
ACS Chem Biol, 9 Nov 2020 DOI: 10.1021/acschembio.0c00477 Link to full text
Abstract: Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate intracellular signaling pathways. One of the red/far-red responsive LID systems, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (SynPCB) and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated the PhyB, PIF, and SynPCB system into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and the PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.

Multichromatic Control of Signaling Pathways in Mammalian Cells.

blue red CRY2/CIB1 PhyB/PIF6 HEK293 Signaling cascade control Multichromatic
Adv Biosyst, 12 Oct 2020 DOI: 10.1002/adbi.202000196 Link to full text
Abstract: The precise control of signaling proteins is a prerequisite to decipher the complexity of the signaling network and to reveal and to study pathways involved in regulating cellular metabolism and gene expression. Optogenetic approaches play an emerging role as they enable the spatiotemporal control of signaling processes. Herein, a multichromatic system is developed by combining the blue light cryptochrome 2 system and the red/far-red light phytochrome B system. The use of three wavelengths allows the orthogonal control of the RAF/ERK and the AKT signaling pathway. Continuous exposure of cells to blue light leads to activation of AKT while simultaneous pulses of red and far-red light enable the modulation of ERK signaling in cells with constantly active AKT signaling. The optimized, orthogonal multichromatic system presented here is a valuable tool to better understand the fine grained and intricate processes involved in cell fate decisions.

Optogenetic control of protein binding using light-switchable nanobodies.

blue red AsLOV2 iLID PhyB/PIF6 HEK293 HEK293T NIH/3T3 Signaling cascade control
Nat Commun, 13 Aug 2020 DOI: 10.1038/s41467-020-17836-8 Link to full text
Abstract: A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.

Optogenetic control of heterologous metabolism in E. coli.

red PhyB/PIF3 E. coli Transgene expression
ACS Synth Biol, 5 Aug 2020 DOI: 10.1021/acssynbio.9b00454 Link to full text
Abstract: Multi-objective optimization of microbial chassis for the production of xenobiotic compounds requires the implementation of metabolic control strategies that permit dynamic distribution of cellular resources between biomass and product formation. We addressed this need in a previous study by engineering the T7 RNA polymerase to be thermally responsive. The modified polymerase is activated only after the temperature of the host cell falls below 18oC, and Escherichia coli cells that employ the protein to transcribe the heterologous lycopene biosynthetic pathway exhibit impressive improvements in productivity. We have expanded our toolbox of metabolic switches in the current study by engineering a version of the T7 RNA polymerase that drives the transition between biomass and product formation upon stimulation with red light. The engineered polymerase is expressed as two distinct polypeptide chains. Each chain comprises one of two photoactive components from Arabidopsis thaliana, phytochrome B (PhyB) and phytochrome-integrating factor 3 (PIF3), as well as the N- or C-terminus domains of both, the vacuolar ATPase subunit (VMA) intein of Saccharomyces cerevisiae and the polymerase. Red light drives photodimerization of PhyB and PIF3, which then brings together the N- and C-terminus domains of the VMA intein. Trans-splicing of the intein follows suit and produces an active form of the polymerase that subsequently transcribes any sequence that is under the control of a T7 promoter. The photodimerization also involves a third element, the cyanobacterial chromophore phycocyanobilin (PCB), which too is expressed heterologously by E. coli. We deployed this version of the T7 RNA polymerase to control the production of lycopene in E. coli and observed tight control of pathway expression. We tested a variety of expression configurations to identify one that imposes the lowest metabolic burden on the strain, and we subsequently optimized key parameters such as the source, moment and duration of photostimulation. We also identified targets for future refinement of the circuit. In summary, our work is a significant advance for the field and greatly expands on previous work by other groups that have used optogenetic circuits to control heterologous metabolism in prokaryotic hosts.

CreLite: An Optogenetically Controlled Cre/loxP System Using Red Light.

red PhyB/PIF6 zebrafish in vivo
Dev Dyn, 3 Aug 2020 DOI: 10.1002/dvdy.232 Link to full text
Abstract: Precise manipulation of gene expression with temporal and spatial control is essential for functional analysis and determining cell lineage relationships in complex biological systems. The Cre-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre. Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. Upon red light illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of the cofactor phycocyanobilin (PCB) to restore Cre activity. Red light exposure of zebrafish embryos harboring a Cre-dependent multi-color fluorescent protein reporter injected with CreLite mRNAs and PCB resulted in Cre activity as measured by the generation of multi-spectral cell labeling in several different tissues. Our data show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different developmental stages, and suggests CreLite may also be useful for temporal and spatial control of gene expression in cell culture, ex vivo organ culture, and other animal models. This article is protected by copyright. All rights reserved.

Optogenetic Rescue of a Patterning Mutant.

red PhyB/PIF6 D. melanogaster in vivo Signaling cascade control Developmental processes
Curr Biol, 9 Jul 2020 DOI: 10.1016/j.cub.2020.06.059 Link to full text
Abstract: Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90 min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.

Optogenetic control of gene expression in plants in the presence of ambient white light.

blue red EL222 PhyB/PIF6 A. thaliana leaf protoplasts N. benthamiana in vivo Transgene expression Multichromatic
Nat Methods, 29 Jun 2020 DOI: 10.1038/s41592-020-0868-y Link to full text
Abstract: Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.

Development of optogenetic tools to manipulate cell cycle checkpoints.

red PhyB/PIF3 S. pombe Cell cycle control
bioRxiv, 23 Jun 2020 DOI: 10.1101/2020.06.22.166264 Link to full text
Abstract: In order to understand the systematic regulation of the cell cycle, we need more precise tools for cell-cycle perturbation. Optogenetics is a powerful technique for precisely controlling cellular signaling at higher spatial and temporal resolution. Here, we report optogenetic tools for the rapid and reversible control of cell-cycle checkpoints with a red/far-red light photoreceptor, phytochrome B (PhyB). We established fission yeast cells producing phycocyanobilin as a chromophore of PhyB, and demonstrated light-dependent protein recruitment to the plasma membrane, nucleus, and kinetochore. Using this system, we developed optogenetic manipulation of the cell cycle in two ways: the Opto-G2/M checkpoint triggered G2/M cell cycle arrest in response to red light, and Opto-SAC induced a spindle assembly checkpoint (SAC) in response to red light and then quickly released the SAC by far-red light.
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