Curated Optogenetic Publication Database

Abstract: The RNA N6-methyladenosine (m6A) modification is a critical regulator of various biological processes, but precise and dynamic control of m6A remains a challenge. In this work, we present a red/far-red light-inducible m6A editing system that enables efficient and reversible modulation of m6A levels with minimal off-target effects. By engineering the CRISPR dCas13 protein and sgRNA with two pairs of light-inducible heterodimerizing proteins, ΔphyA/FHY1 and Bphp1/PspR2, we achieved targeted recruitment of m6A effectors. This system significantly enhances m6A writing efficiency and allows dynamic regulation of m6A deposition and removal on specific transcripts, such as SOX2 and ACTB. Notably, reversible m6A editing was achieved through cyclic modulation at a single target site, demonstrating the ability to influence mRNA expression and modulate the differentiation state of human embryonic stem cells. This optogenetic platform offers a precise, versatile tool for cyclic and reversible m6A regulation, with broad implications for understanding RNA biology and its potential applications in research and medicine.

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: Synthetic biology applications require finely tuned gene expression, often mediated by synthetic transcription factors (sTFs) compatible with the human genome and transcriptional regulation mechanisms. While various DNA-binding and activation domains have been developed for different applications, advanced artificially controllable sTFs with improved regulatory capabilities are required for increasingly sophisticated applications. Here, in mammalian cells and mice, we validate the transactivator function and homo-/heterodimerization activity of the plant-derived phytochrome chaperone proteins, FHY1 and FHL. Our results demonstrate that FHY1/FHL form a photosensing transcriptional regulation complex (PTRC) through interaction with the phytochrome, ΔPhyA, that can toggle between active and inactive states through exposure to red or far-red light, respectively. Exploiting this capability, we develop a light-switchable platform that allows for orthogonal, modular, and tunable control of gene transcription, and incorporate it into a PTRC-controlled CRISPRa system (PTRCdcas) to modulate endogenous gene expression. We then integrate the PTRC with small molecule- or blue light-inducible regulatory modules to construct a variety of highly tunable systems that allow rapid and reversible control of transcriptional regulation in vitro and in vivo. Validation and deployment of these plant-derived phytochrome chaperone proteins in a PTRC platform have produced a versatile, powerful tool for advanced research and biomedical engineering applications.

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.

Abstract: Optogenetic technologies have transformed our ability to precisely control biological processes in time and space. Yet, current eukaryotic optogenetic systems are limited by large or complex optogenetic modules, long illumination times, low tissue penetration or slow activation and deactivation kinetics. Here, we report a red/far-red light-mediated and miniaturized Δphytochrome A (ΔPhyA)-based photoswitch (REDMAP) system based on the plant photoreceptor PhyA, which rapidly binds the shuttle protein far-red elongated hypocotyl 1 (FHY1) under illumination with 660-nm light with dissociation occurring at 730 nm. We demonstrate multiple applications of REDMAP, including dynamic on/off control of the endogenous Ras/Erk mitogen-activated protein kinase (MAPK) cascade and control of epigenetic remodeling using a REDMAP-mediated CRISPR-nuclease-deactivated Cas9 (CRISPR-dCas9) (REDMAPcas) system in mice. We also demonstrate the utility of REDMAP tools for in vivo applications by activating the expression of transgenes delivered by adeno-associated viruses (AAVs) or incorporated into cells in microcapsules implanted into mice, rats and rabbits illuminated by light-emitting diodes (LEDs). Further, we controlled glucose homeostasis in type 1 diabetic (T1D) mice and rats using REDMAP to trigger insulin expression. REDMAP is a compact and sensitive tool for the precise spatiotemporal control of biological activities in animals with applications in basic biology and potentially therapy.

Abstract: Advances in synthetic biology will require spatio-temporal regulation of biological processes in heterologous host cells. We develop a light-switchable, two-hybrid interaction in yeast, based upon the Arabidopsis proteins PHYTOCHROME A and FAR-RED ELONGATED HYPOCOTYL 1-LIKE. Light input to this regulatory module allows dynamic control of a light-emitting LUCIFERASE reporter gene, which we detect by real-time imaging of yeast colonies on solid media.