Curated Optogenetic Publication Database

Abstract: Uveal melanoma is the most common intraocular primary malignancy in adults and has been considered a fatal disease for decades. Optogenetics is an emerging technique that can control the activation of signaling components via irradiation with visible light. The clinical translation of optogenetics has been limited because of the need for surgical implantation of electrodes and relatively shallow tissue penetration. As visible light easily penetrates the eyes, we hypothesized that an optogenetics approach can be an effective treatment of uveal melanoma without surgery. In this study, we evaluated the feasibility of this strategy by using a genetically encoded optogenetic system based on reversible blue light-induced binding pairs between Fas-CIB1-EGFP and CRY2-mCherry-FADD. Subretinal injection of B16 cells was performed to create a uveal melanoma model. Plasmids pairs were co-transfected into B16 cells. We found that blue light irradiation dynamically controlled the translocation of FADD to Fas on the plasma membrane and induced the apoptosis of B16 cells transfected with the optogenetic nanosystem in vitro. Moreover, the blue light-controlled optogenetic nanosystem suppressed the growth of uveal melanoma in vivo by inducing apoptosis. These results suggest that light-controlled optogenetic therapy can be used as a potential novel therapeutic strategy for uveal melanoma.

Abstract: Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.

Abstract: Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Förster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2. Based on structure-guided engineering, a chimeric protein with 40% FRET efficiency was developed and named 2P-activatable CRY2, or 2paCRY2. 2paCRY2 was employed to develop a RAF1 activation system named 2paRAF. In three-dimensionally cultured cells expressing 2paRAF, extracellular signal-regulated kinase (ERK) was efficiently activated by 2P excitation at single-cell resolution. Photoactivation of ERK was also accomplished in the epidermal cells of 2paRAF-expressing mice. We further developed an mTFP1-fused LOV domain that exhibits efficient response to 2P excitation. Collectively, FRAPA will pave the way to single-cell optical control of signaling pathways in vivo.

Abstract: The CRISPR-Cpf1 endonuclease has recently been demonstrated as a powerful tool to manipulate targeted gene sequences. Here, we performed an extensive screening of split Cpf1 fragments and identified a pair that, combined with inducible dimerization domains, enables chemical- and light-inducible genome editing in human cells. We also identified another split Cpf1 pair that is spontaneously activated. The newly generated amino and carboxyl termini of the spontaneously activated split Cpf1 can be repurposed as de novo fusion sites of artificial effector domains. Based on this finding, we generated an improved split dCpf1 activator, which has the potential to activate endogenous genes more efficiently than a previously established dCas9 activator. Finally, we showed that the split dCpf1 activator can efficiently activate target genes in mice. These results demonstrate that the present split Cpf1 provides an efficient and sophisticated genome manipulation in the fields of basic research and biotechnological applications.

Abstract: Abstract not available.

Abstract: A common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.

Abstract: Light in the near-infrared optical window (NIRW) penetrates deep through mammalian tissues, including the skull and brain tissue. Here we engineered an adenylate cyclase (AC) activated by NIRW light (NIRW-AC) and suitable for mammalian applications. To accomplish this goal, we constructed fusions of several bacteriophytochrome photosensory and bacterial AC modules using guidelines for designing chimeric homodimeric bacteriophytochromes. One engineered NIRW-AC, designated IlaM5, has significantly higher activity at 37 °C, is better expressed in mammalian cells, and can mediate cAMP-dependent photoactivation of gene expression in mammalian cells, in favorable contrast to the NIRW-ACs engineered earlier. The ilaM5 gene expressed from an AAV vector was delivered into the ventral basal thalamus region of the mouse brain, resulting in the light-controlled suppression of the cAMP-dependent wave pattern of the sleeping brain known as spindle oscillations. Reversible spindle oscillation suppression was observed in sleeping mice exposed to light from an external light source. This study confirms the robustness of principles of homodimeric bacteriophytochrome engineering, describes a NIRW-AC suitable for mammalian optogenetic applications, and demonstrates the feasibility of controlling brain activity via NIRW-ACs using transcranial irradiation.

Abstract: Mononegaviruses are promising tools as oncolytic vectors and transgene delivery vectors for gene therapy and regenerative medicine. By using the Magnet proteins, which reversibly heterodimerize upon blue light illumination, photocontrollable mononegaviruses (measles and rabies viruses) were generated. The Magnet proteins were inserted into the flexible domain of viral polymerase, and viruses showed strong replication and oncolytic activities only when the viral polymerases were activated by blue light illumination.

Abstract: Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.

Abstract: In vivo noninvasively manipulating biological functions by the mediation of biosafe near infrared (NIR) light is becoming increasingly popular. For these applications, upconversion rare-earth nanomaterial holds great promise as a novel photonic element, and has been widely adopted in optogenetics. In this article, an upconversion optogenetic nanosystem that was promised to achieve autophagy up-regulation with spatiotemporal precision was designed. The implantable, wireless, recyclable, less-invasive and biocompatible system worked via two separated parts: blue light-receptor optogenetics-autophagy upregulation plasmids, for protein import; upconversion rods-encapsulated flexible capsule (UCRs-capsule), for converting tissue-penetrative NIR light into local visible blue light. Results validated that this system could achieve up-regulation of autophagy in vitro (in both HeLa and 293T cell lines) and remotely penetrate tissue (∼3.5 mm) in vivo. Since autophagy serves at a central position in intracellular signalling pathways, which is correlative with diverse pathologies, we expect that this method could establish an upconversion material-based autophagy up-regulation strategy for fundamental and clinical applications.

Abstract: Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.

Abstract: Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this 'PAC-K' silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.

Abstract: Gene expression and its network structure are dynamically altered in multicellular systems during morphological, functional, and pathological changes. To precisely analyze the functional roles of dynamic gene expression changes, tools that manipulate gene expression at fine spatiotemporal resolution are needed. The tetracycline (Tet)-controlled gene expression system is a reliable drug-inducible method, and it is used widely in many mammalian cultured cells and model organisms. Here, we develop a photoactivatable (PA)-Tet-OFF/ON system for precise temporal control of gene expression at single-cell resolution. By integrating the cryptochrome 2-cryptochrome-interacting basic helix-loop-helix 1 (Cry2-CIB1) light-inducible binding switch, expression of the gene of interest is tightly regulated under the control of light illumination and drug application in our PA-Tet-OFF/ON system. This system has a large dynamic range of downstream gene expression and rapid activation/deactivation kinetics. We also demonstrate the optogenetic regulation of exogenous gene expression in vivo, such as in developing and adult mouse brains.

Abstract: The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.

Abstract: EphB2 is involved in enhancing synaptic transmission and gene expression. To explore the roles of EphB2 in memory formation and enhancement, we used a photoactivatable EphB2 (optoEphB2) to activate EphB2 forward signaling in pyramidal neurons in lateral amygdala (LA). Photoactivation of optoEphB2 during fear conditioning, but not minutes afterward, enhanced long-term, but not short-term, auditory fear conditioning. Photoactivation of optoEphB2 during fear conditioning led to activation of the cAMP/Ca2+ responsive element binding (CREB) protein. Application of light to a kinase-dead optoEphB2 in LA did not lead to enhancement of long-term fear conditioning memory or to activation of CREB. Long-term, but not short-term, auditory fear conditioning memory was impaired in mice lacking EphB2 forward signaling (EphB2lacZ/lacZ). Activation of optoEphB2 in LA of EphB2lacZ/lacZ mice enhanced long-term fear conditioning memory. The present findings show that the level of EphB2 forward signaling activity during learning determines the strength of long-term memory consolidation.

Abstract: Near-infrared (NIR, 740-780 nm) optogenetic systems are well-suited to spectral multiplexing with blue-light-controlled tools. Here, we present two protocols, one for regulation of gene transcription and another for control of protein localization, that use a NIR-responsive bacterial phytochrome BphP1-QPAS1 optogenetic pair. In the first protocol, cells are transfected with the optogenetic constructs for independently controlling gene transcription by NIR (BphP1-QPAS1) and blue (LightOn) light. The NIR and blue-light-controlled gene transcription systems show minimal spectral crosstalk and induce a 35- to 40-fold increase in reporter gene expression. In the second protocol, the BphP1-QPAS1 pair is combined with a light-oxygen-voltage-sensing (LOV) domain-based construct into a single optogenetic tool, termed iRIS. This dual-light-controllable protein localization tool allows tridirectional protein translocation among the cytoplasm, nucleus and plasma membrane. Both procedures can be performed within 3-5 d. Use of NIR light-controlled optogenetic systems should advance basic and biomedical research.

Abstract: Gene therapy is expected to be utilized for the treatment of various diseases. However, the spatiotemporal resolution of current gene therapy technology is not high enough. In this study, we generated a new technology for spatiotemporally controllable gene therapy. We introduced optogenetic and CRISPR/Cas9 techniques into a recombinant adenovirus (Ad) vector, which is widely used in clinical trials and exhibits high gene transfer efficiency, to generate an illumination-dependent spatiotemporally controllable gene regulation system (designated the Opt/Cas-Ad system). We generated an Opt/Cas-Ad system that could regulate a potential tumor suppressor gene, and we examined the effectiveness of this system in cancer treatment using a xenograft tumor model. With the Opt/Cas-Ad system, highly selective tumor treatment could be performed by illuminating the tumor. In addition, Opt/Cas-Ad system-mediated tumor treatment could be stopped simply by turning off the light. We believe that our Opt/Cas-Ad system can enhance both the safety and effectiveness of gene therapy.

Abstract: In vivo the application of optogenetic manipulation in deep tissue is seriously obstructed by the limited penetration depth of visible light that is continually applied to activate a photoactuator. Herein, we designed a versatile upconversion optogenetic nanosystem based on a blue-light-mediated heterodimerization module and rare-earth upconversion nanoparticles (UCNs). The UCNs worked as a nanotransducer to convert external deep-tissue-penetrating near-infrared (NIR) light to local blue light to noninvasively activate photoreceptors for optogenetic manipulation in vivo. In this, we demonstrated that deeply penetrating NIR light could be used to control the apoptotic signaling pathway of cancer cells in both mammalian cells and mice by UCNs. We believe that this interesting NIR-light-responsive upconversion optogenetic nanotechnology has significant application potentials for both basic research and clinical applications in vivo.

Abstract: The precise spatial and temporal control of gene expression, cell differentiation, and tissue morphogenesis has widespread application in regenerative medicine and the study of tissue development. In this work, we applied optogenetics to control cell differentiation and new tissue formation. Specifically, we engineered an optogenetic "on" switch that provides permanent transgene expression following a transient dose of blue light illumination. To demonstrate its utility in controlling cell differentiation and reprogramming, we incorporated an engineered form of the master myogenic factor MyoD into this system in multipotent cells. Illumination of cells with blue light activated myogenic differentiation, including upregulation of myogenic markers and fusion into multinucleated myotubes. Cell differentiation was spatially patterned by illumination of cell cultures through a photomask. To demonstrate the application of the system to controlling in vivo tissue development, the light inducible switch was used to control the expression of VEGF and angiopoietin-1, which induced angiogenic sprouting in a mouse dorsal window chamber model. Live intravital microscopy showed illumination-dependent increases in blood-perfused microvasculature. This optogenetic switch is broadly useful for applications in which sustained and patterned gene expression is desired following transient induction, including tissue engineering, gene therapy, synthetic biology, and fundamental studies of morphogenesis.

Abstract: Despite recent advances in optogenetics, it remains challenging to manipulate gene expression in specific populations of neurons. We present a dual-protein switch system, Cal-Light, that translates neuronal-activity-mediated calcium signaling into gene expression in a light-dependent manner. In cultured neurons and brain slices, we show that Cal-Light drives expression of the reporter EGFP with high spatiotemporal resolution only in the presence of both blue light and calcium. Delivery of the Cal-Light components to the motor cortex of mice by viral vectors labels a subset of excitatory and inhibitory neurons related to learned lever-pressing behavior. By using Cal-Light to drive expression of the inhibitory receptor halorhodopsin (eNpHR), which responds to yellow light, we temporarily inhibit the lever-pressing behavior, confirming that the labeled neurons mediate the behavior. Thus, Cal-Light enables dissection of neural circuits underlying complex mammalian behaviors with high spatiotemporal precision.

Abstract: Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

Abstract: With the increasingly dominant role of smartphones in our lives, mobile health care systems integrating advanced point-of-care technologies to manage chronic diseases are gaining attention. Using a multidisciplinary design principle coupling electrical engineering, software development, and synthetic biology, we have engineered a technological infrastructure enabling the smartphone-assisted semiautomatic treatment of diabetes in mice. A custom-designed home server SmartController was programmed to process wireless signals, enabling a smartphone to regulate hormone production by optically engineered cells implanted in diabetic mice via a far-red light (FRL)-responsive optogenetic interface. To develop this wireless controller network, we designed and implanted hydrogel capsules carrying both engineered cells and wirelessly powered FRL LEDs (light-emitting diodes). In vivo production of a short variant of human glucagon-like peptide 1 (shGLP-1) or mouse insulin by the engineered cells in the hydrogel could be remotely controlled by smartphone programs or a custom-engineered Bluetooth-active glucometer in a semiautomatic, glucose-dependent manner. By combining electronic device-generated digital signals with optogenetically engineered cells, this study provides a step toward translating cell-based therapies into the clinic.

Abstract: Few tools exist to visualize and manipulate neurons that are targets of neuromodulators. We present iTango, a light- and ligand-gated gene expression system based on a light-inducible split tobacco etch virus protease. Cells expressing the iTango system exhibit increased expression of a marker gene in the presence of dopamine and blue-light exposure, both in vitro and in vivo. We demonstrated the iTango system in a behaviorally relevant context, by inducing expression of optogenetic tools in neurons under dopaminergic control during a behavior of interest. We thereby gained optogenetic control of these behaviorally relevant neurons. We applied the iTango system to decipher the roles of two classes of dopaminergic neurons in the mouse nucleus accumbens in a sensitized locomotor response to cocaine. Thus, the iTango platform allows for control of neuromodulatory circuits in a genetically and functionally defined manner with spatial and temporal precision.

Abstract: Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1-2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ∼1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.

Abstract: Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (∼0.04 W m(-2)) or short periods of pulsed illumination (∼30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.