Curated Optogenetic Publication Database

Abstract: Evolving dynamic, multi-state, and computational protein functionalities is challenging because it requires selection pressure on all the states of a protein of interest (POI) and the transitions between them. To create a continuous directed evolution paradigm for such properties, we genetically engineered budding yeast for optogenetic input to switch a POI "on" and "off," which, in turn, controls a Cdk1 cyclin that is essential for one cell-cycle stage but detrimental for another. The method, "optovolution," generates dynamic selection pressure on POI cycling at the timescale of tens of minutes. We used it to evolve 19 new variants of the LOV transcription factor El222, including in vivo green-light-responsive variants allowing LOV color-multiplexing. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing phycocyanobilin (PCB) unnecessary. Finally, we demonstrated the generality of the method by evolving a non-light-responsive AND gate (PEST-rtTA). Optovolution makes difficult-to-engineer protein functionalities continuously evolvable.

Abstract: The genome folds inside the cell nucleus into hierarchical architectural features, such as chromatin loops and domains. If and how this genome organization influences the regulation of gene expression remains only partially understood. The structure-function relationship of genomes has traditionally been probed by population-wide measurements after mutation of crucial DNA elements or by perturbation of chromatin-associated proteins. To circumvent possible pleiotropic effects of such approaches, we have developed OptoLoop, an optogenetic system that allows direct manipulation of chromatin contacts by light in a controlled fashion. OptoLoop is based on the fusion between a nuclease-dead SpCas9 protein and the light-inducible oligomerizing protein CRY2. We demonstrate that OptoLoop can bring together genomically distant, repetitive DNA loci. As a proof-of-principle application of OptoLoop, we probed the functional role of DNA looping in the regulation of the human telomerase gene TERT. By analyzing the extent of chromatin looping and nascent RNA production at individual alleles, we find evidence for looping-mediated repression of TERT. In sum, OptoLoop represents a novel means for the interrogation of structure-function relationships in the genome.

Abstract: Bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a crucial role in bacterial signaling pathways, allowing bacterial cells to respond to various environmental stimuli. The prevalence of c-di-GMP and its potential applications underscore the necessity for developing tools and methods to regulate intracellular c-di-GMP levels. Optogenetic control of c-di-GMP dynamics is particularly attractive because it enables tunable and spatiotemporal regulation of c-di-GMP metabolism. The development of sensitive optogenetic control systems requires highly active, light-responsive c-di-GMP synthases. Here, we report an engineered, highly active photosensitive c-di-GMP synthase, BphS-13. This engineered c-di-GMP synthase was developed from a near-infrared (NIR) light-activable bacteriophytochrome c-di-GMP synthase, BphS, using a three-step directed evolution process that included error-prone PCR, in vitro homologous recombination, and site-directed mutagenesis. After two rounds of this directed evolution strategy, we generated a BphS variant with 13 mutations, referred to as BphS-13. The diguanylate cyclase (DGC) activity of BphS-13 was approximately 13 times higher than that of the original BphS, and it exhibited tightly regulated DGC activity in response to NIR light with minimal leakage in the dark. We then demonstrated the effectiveness of BphS-13 in controlling biofilm dynamics. Overall, this study highlights BphS-13 as a highly active and photosensitive tool for optogenetic applications in biotechnology and suggests its future potential application in mammalian systems for precise control of gene expression, particularly given the lack of native c-di-GMP signaling pathways in mammalian cells.

Abstract: How do cells maintain robust, yet flexible polarization for directed motion? Recent optogenetic experiments by Town and Weiner on neutrophil-like HL-60 cells strongly point to the essential role of a Rac-inhibitor (downstream of the small GTPase Rac) in shaping requisite negative feedback that allows cells to respond to rapidly changing directional cues. Here we adapt a previous mathematical model for cell polarity to model interactions of Rac, its putative inhibitor, and upstream PIP3 (a product of the optogenetically stimulated PI3K). We fit parameters in our partial differential equation (PDE) model to temporal and spatial experimental data. Cell shapes, motility, and stimulus responses are modeled in 2D simulations, with PDEs solved along the cell edge. We show that the Rac-inhibitor-PIP3 circuit accounts for the optogenetic data (including exotic cell trajectories), that it is the minimal circuit to do so, and that it improves gradient sensing under noisy or dynamic conditions.

Abstract: The robustness and broad applicability of an optogenetic tool depends heavily on the properties of the underlying photoreceptor protein and its cognate binding partner - the light responsive ‘core’. Current red light optogenetic systems for use in mammalian cells all rely on phytochrome based photoreceptors. These are large (70 kDa) proteins that act as dimers, thereby enforce dimerization on attached proteins. Naturally occurring or engineered binding partners can function effectively in certain cases, but large size, complex mode of interaction, background binding, relatively weak affinity and/or low fold changes between on and off states are significant limitations. Using structure-based design and directed evolution we developed a small (17 kDa) monomeric bilverdin binding photoreceptor FenixS, and a highly selective, high-affinity binder, Ash1 (6 kDa). Negligible off-state binding and a >1200-fold increase in binding affinity upon 700 nm illumination result in a high performance, ultra-low background, light responsive core for a diverse range of applications. An optogenetic tool for red light activation of transcription in mammalian cells based on the FenixS-Ash1 core exhibits robust performance without the need for biliverdin supplementation.

Abstract: Optogenetics has emerged as a powerful tool for regulating cellular processes due to its noninvasive nature and precise spatiotemporal control. Far-red light (FRL) has increasingly been used in the optogenetic control of mammalian cells due to its low toxicity and high tissue penetration. However, robust and orthogonal FRL sensors are lacking in bacteria. Here, we established an orthogonal FRL sensor in Escherichia coli with a maximum dynamic range exceeding 230-fold based on the RfpA-RfpC-RfpB (RfpABC) signaling system that regulates the far-red light photoacclimation (FaRLiP) in cyanobacteria. We identified a conserved DNA motif in the promoter sequences of the Chl f synthase gene and other genes in the FaRLiP gene clusters, termed the far-red light-regulatory (FLR) motif, which enables the light-responsive activation of gene expression through its interaction with RfpB. Based on the FLR motif, we simplified the FLR-containing promoters and characterized their activation abilities and dynamic ranges, which can be utilized in different synthetic biology scenarios. Additionally, one or two FLR motifs are present at other loci within the FaRLiP gene cluster, providing further FRL-inducible promoter resources. The FRL sensor exhibits effective activation and suppression under low-intensity FRL and white light, respectively, and remains functional in darkness. In conclusion, this study advances the understanding of the regulatory mechanisms of FaRLiP in cyanobacteria and provides robust and orthogonal FRL sensors for synthetic biology applications.

Abstract: In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light-responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome-based hydrogels with an increased dynamic range for the storage modulus but a different light-response for the loss modulus compared to the original Cph1-based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1-R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems.

Abstract: Methods to acutely manipulate protein interactions at the subcellular level are powerful tools in cell biology. Several blue-light-dependent optical dimerization tools have been developed. In these systems one protein component of the dimer (the bait) is directed to a specific subcellular location, while the other component (the prey) is fused to the protein of interest. Upon illumination, binding of the prey to the bait results in its subcellular redistribution. Here, we compared and quantified the extent of light-dependent dimer occurrence in small, subcellular volumes controlled by three such tools: Cry2/CIB1, iLID, and Magnets. We show that both the location of the photoreceptor protein(s) in the dimer pair and its (their) switch-off kinetics determine the subcellular volume where dimer formation occurs and the amount of protein recruited in the illuminated volume. Efficient spatial confinement of dimer to the area of illumination is achieved when the photosensitive component of the dimerization pair is tethered to the membrane of intracellular compartments and when on and off kinetics are extremely fast, as achieved with iLID or Magnets. Magnets and the iLID variants with the fastest switch-off kinetics induce and maintain protein dimerization in the smallest volume, although this comes at the expense of the total amount of dimer. These findings highlight the distinct features of different optical dimerization systems and will be useful guides in the choice of tools for specific applications.

Abstract: Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.

Abstract: Optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein-protein interactions. Such tools have been useful for regulating cellular pathways and processes with high spatiotemporal resolution in live cells, and a growing number of dimerizer systems are available. As these systems have been characterized by different groups using different methods, it has been difficult for users to compare their properties. Here, we set about to systematically benchmark the properties of four optical dimerizer systems, CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6. Using a yeast transcriptional assay, we find significant differences in light sensitivity and fold-activation levels between the red light regulated systems but similar responses between the CRY2/CIB and TULIP systems. Further comparison of the ability of the CRY2/CIB1 and TULIP systems to regulate a yeast MAPK signaling pathway also showed similar responses, with slightly less background activity in the dark observed with CRY2/CIB. In the process of developing this work, we also generated an improved blue-light-regulated transcriptional system using CRY2/CIB in yeast. In addition, we demonstrate successful application of the CRY2/CIB dimerizers using a membrane-tethered CRY2, which may allow for better local control of protein interactions. Taken together, this work allows for a better understanding of the capacities of these different dimerization systems and demonstrates new uses of these dimerizers to control signaling and transcription in yeast.