Curated Optogenetic Publication Database

Abstract: The Cre-loxP recombination system enables precise genome engineering; however, existing photoactivatable Cre tools suffer from several limitations, including low DNA recombination efficiency, background activation, slow activation kinetics, and poor tissue penetration. Here, we present REDMAPCre, a red-light-controlled split-Cre system based on the ΔPhyA/FHY1 interaction. REDMAPCre enables rapid activation (1-s illumination) and achieves an 85-fold increase in reporter expression over background levels. We demonstrate its efficient regulation of DNA recombination in mammalian cells and mice, as well as its compatibility with other inducible recombinase systems for Boolean logic-gated DNA recombination. Using a single-vector adeno-associated virus delivery system, we successfully induced REDMAPCre-mediated DNA recombination in mice. Furthermore, we generated a REDMAPCre transgenic mouse line and validated its efficient, light-dependent recombination across multiple organs. To explore its functional applications, REDMAPCre transgenic mice were crossed with isogenic Cre-dependent reporter mice, enabling optogenetic induction of insulin resistance and hepatic lipid accumulation via Cre-dependent overexpression of ubiquitin-like with PHD and RING finger domains 1 (UHRF1), as well as targeted cell ablation through diphtheria toxin fragment A expression. Collectively, REDMAPCre provides a powerful tool for achieving remote control of recombination and facilitating functional genetic studies in living systems.

Abstract: Induced pluripotent stem cells (iPSCs) share similar cellular features and various antigens profiles with cancer cells. Leveraging these characteristics, iPSCs hold great promise for developing wide-spectrum vaccines against cancers. In practice, iPSCs are typically combined with immune adjuvants to enhance antitumor immune responses; however, traditional adjuvants lack controllability and can induce systemic toxicity, which has limited their broad application. Here, a red/far-red light-controlled iPSC-based vaccine (RIVA) based on the chimeric photosensory protein FnBphP and its interaction partner LDB3 is developed; RIVA preserves the intrinsic tumor antigens of iPSCs and enables optogenetic control of an immune adjuvant's (IFN-β) expression under red light illumination. Experiments in multiple mouse tumor models demonstrate that RIVA inhibits tumor growth and improves animal survival in prophylactic and therapeutic settings, including against pulmonary metastatic 4T1 breast cancer. RIVA efficiently stimulates dendritic cell maturation, eliciting innate immune activation effects through NK cells and elicit adaptive immune anti-tumor responses through CD4+ and CD8+ T cells. Moreover, RIVA protects animals against tumor re-challenge by inducing strong immunological memory, with minimal systemic toxicity. This study demonstrates RIVA as an effective optogenetic approach for developing safe multi-antigen vaccines for the prevention and treatment of cancer.

Abstract: Bacteria-based therapies hold great promise for cancer treatment due to their selective tumor colonization and proliferation. However, clinical application is hindered by the need for safe, precise control systems to regulate local therapeutic payload expression and release. Here we developed a near-infrared (NIR) light-mediated PadC-based photoswitch (NETMAP) system based on a chimeric phytochrome-activated diguanylyl cyclase (PadC) and a cyclic diguanylate monophosphate-dependent transcriptional activator (MrkH). The NETMAP-engineered bacteria exhibited antitumor performance in mouse tumor models with different levels of immunogenicity. Specifically, in immunogenic lymphoma tumors, NIR-induced PD-L1 and CTLA-4 nanobodies enhanced the activation of adaptive immunity. In low-immunogenic tumors-including mouse-derived colon cancer models, an orthotopic human breast cancer cell line-derived xenograft model and a colorectal cancer patient-derived xenograft model-NIR-induced azurin and cytolysin A predominantly led to tumor inhibition. Our study identifies an NIR light-mediated therapeutic platform for engineered bacteria-based therapies with customizable outputs and precise dosage 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: Diabetes treatment and rehabilitation are usually a lifetime process. Optogenetic engineered designer cell-therapy holds great promise in regulating blood glucose homeostasis. However, portable, sustainable, and long-term energy supplementation has previously presented a challenge for the use of optogenetic stimulation in vivo. Herein, we purpose a self-powered optogenetic system (SOS) for implantable blood glucose control. The SOS consists of a biocompatible far-red light (FRL) source, FRL-triggered transgene-expressing cells, a power management unit, and a flexible implantable piezoelectric nanogenerator (i-PENG) to supply long-term energy by converting biomechanical energy into electricity. Our results show that this system can harvest energy from body movement and power the FRL source, which then significantly enhanced production of a short variant of human glucagon-like peptide 1 (shGLP-1) in vitro and in vivo. Indeed, diabetic mice equipped with the SOS showed rapid restoration of blood glucose homeostasis, improved glucose, and insulin tolerance. Our results suggest that the SOS is sufficiently effective in self-powering the modulation of therapeutic outputs to control glucose homeostasis and, furthermore, present a new strategy for providing energy in optogenetic-based cell therapy.

Abstract: The CRISPR-Cas12a has been harnessed as a powerful tool for manipulating targeted gene expression. The possibility to manipulate the activity of CRISPR-Cas12a with a more precise spatiotemporal resolution and deep tissue permeability will enable targeted genome engineering and deepen our understanding of the gene functions underlying complex cellular behaviors. However, currently available inducible CRISPR-Cas12a systems are limited by diffusion, cytotoxicity, and poor tissue permeability. Here, we developed a far-red light (FRL)–inducible CRISPR-Cas12a (FICA) system that can robustly induce gene editing in mammalian cells, and an FRL-inducible CRISPR-dCas12a (FIdCA) system based on the protein-tagging system SUperNova (SunTag) that can be used for gene activation under light-emitting diode–based FRL. Moreover, we show that the FIdCA system can be deployed to activate target genes in mouse livers. These results demonstrate that these systems developed here provide robust and efficient platforms for programmable genome manipulation in a noninvasive and spatiotemporal fashion.

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: Diabetes affects almost half a billion people, and all individuals with type 1 diabetes (T1D) and a large portion of individuals with type 2 diabetes rely on self-administration of the peptide hormone insulin to achieve glucose control. However, this treatment modality has cumbersome storage and equipment requirements and is susceptible to fatal user error. Here, reasoning that a cell-based therapy could be coupled to an external induction circuit for blood glucose control, as a proof of concept we developed far-red light (FRL)-activated human islet-like designer (FAID) cells and demonstrated how FAID cell implants achieved safe and sustained glucose control in diabetic model mice. Specifically, by introducing a FRL-triggered optogenetic device into human mesenchymal stem cells (hMSCs), which we encapsulated in poly-(l-lysine)-alginate and implanted subcutaneously under the dorsum of T1D model mice, we achieved FRL illumination-inducible secretion of insulin that yielded improvements in glucose tolerance and sustained blood glucose control over traditional insulin glargine treatment. Moreover, the FAID cell implants attenuated both oxidative stress and development of multiple diabetes-related complications in kidneys. This optogenetics-controlled "living cell factory" platform could be harnessed to develop multiple synthetic designer therapeutic cells to achieve long-term yet precisely controllable drug delivery.