Showing 1 - 14 of 14 results
Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex.
Human hepatic C3A cells have been applied in bioartificial liver development, although these cells display low intrinsic cytochrome P450 3A4 (CYP3A4) enzyme activity. We aimed to enhance CYP3A4 enzyme activity of C3A cells utilizing CRISPR gene editing technology. We designed two CYP3A4 expression enhanced systems applying clustered regularly interspaced short palindromic repeats (CRISPR) gene technology: a CRISPR-on activation system including dCas9-VP64-GFP and two U6-sgRNA-mCherry elements, and a light-controlled CRISPR-on activation system combining our CRISPR-on activation system with an optical control system to facilitate regulation of CYP3A4 expression for various applications. Results of enzymatic activity assays displayed increased CYP3A4 activity in C3A cells expressing the CRISPR-on activation system compared with C3A cells. In addition, CYP3A4 activity increased in C3A cells expressing the light-controlled CRISPR-on activation system under blue light radiation compared with C3A cells. Notably, there was no statistical difference in the increase of CYP3A4 protein amounts induced by these two methods. After expansion in culture, C3A cells with the light-controlled CRISPR-on activation system exhibited no statistical difference in CYP3A4 mRNA levels between generations. Our findings provide a method to stably enhance functional gene expression in bioartificial liver cells with the potential for large-scale cell expansion.
Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9.
Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.
Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation.
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.
Rewiring Calcium Signaling for Precise Transcriptional Reprogramming.
Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca2+-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.
Generation of Optogenetically Modified Adenovirus Vector for Spatiotemporally Controllable Gene Therapy.
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.
Modulation of cyclic nucleotide-mediated cellular signaling and gene expression using photoactivated adenylyl cyclase as an optogenetic tool.
Cyclic nucleotide signaling pathway plays a significant role in various biological processes such as cell growth, transcription, inflammation, in microbial pathogenesis, etc. Modulation of cyclic nucleotide levels by optogenetic tools has overcome certain limitations of studying transduction cascade by pharmacological agents and has allowed several ways to modulate biological processes in a spatiotemporal manner. Here, we have shown the optogenetic modulation of the cyclooxygenase 2 (Cox-2) gene expression and their downstream effector molecule (PGE2) in HEK-293T cells and the development process of Dictyostelium discoideum via modulating the cyclic nucleotide (cAMP) signaling pathway utilizing photoactivated adenylyl cyclases (PACs) as an optogenetic tool. Light-induced activation of PACs in HEK-293T cells increases the cAMP level that leads to activation of cAMP response element-binding protein (CREB) transcription factor and further upregulates downstream Cox-2 gene expression and their downstream effector molecule prostaglandin E2. In D. discoideum, the light-regulated increase in cAMP level affects the starvation-induced developmental process. These PACs could modulate the cAMP levels in a light-dependent manner and have a potential to control gene expression and their downstream effector molecules with varying magnitude. It would enable one to utilize PAC as a tool to decipher cyclic nucleotide mediated signaling pathway regulations and their mechanism.
CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation.
Our improved CRISPR-Cas9-based photoactivatable transcription systems, CPTS2.0 and Split-CPTS2.0, enable high blue-light-inducible activation of endogenous target genes in various human cell lines. We achieved reversible activation of target genes with CPTS2.0 and induced neuronal differentiation in induced pluripotent stem cells (iPSCs) by upregulating NEUROD1 with Split-CPTS2.0.
Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo.
Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. Here, we use optogenetic manipulation to switch off Bicoid-dependent transcription in the early Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. We find that Bicoid transcriptional activity is dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of Bicoid decoding.
Optogenetic control of nuclear protein export.
Active nucleocytoplasmic transport is a key mechanism underlying protein regulation in eukaryotes. While nuclear protein import can be controlled in space and time with a portfolio of optogenetic tools, protein export has not been tackled so far. Here we present a light-inducible nuclear export system (LEXY) based on a single, genetically encoded tag, which enables precise spatiotemporal control over the export of tagged proteins. A constitutively nuclear, chromatin-anchored LEXY variant expands the method towards light inhibition of endogenous protein export by sequestering cellular CRM1 receptors. We showcase the utility of LEXY for cell biology applications by regulating a synthetic repressor as well as human p53 transcriptional activity with light. LEXY is a powerful addition to the optogenetic toolbox, allowing various novel applications in synthetic and cell biology.
Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor.
Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.
Optogenetic Inhibitor of the Transcription Factor CREB.
Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events.
A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.
Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR-Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.
CRISPR-Cas9-based photoactivatable transcription system.
Targeted endogenous gene activation is necessary for understanding complex gene networks and has great potential in medical and industrial applications. The CRISPR-Cas system offers simple and powerful tools for this purpose. However, these CRISPR-Cas-based tools for activating user-defined genes are unable to offer precise temporal control of gene expression, despite the fact that many biological phenomena are regulated by highly dynamic patterns of gene expression. Here we created a light-inducible, user-defined, endogenous gene activation system based on CRISPR-Cas9. We demonstrated that this CRISPR-Cas9-based transcription system can allow rapid and reversible targeted gene activation by light. In addition, using this system, we have exemplified photoactivation of multiple user-defined endogenous genes in mammalian cells. The present CRISPR-Cas9-based transcription system offers simple and versatile approaches for precise endogenous gene activation in basic biological research and biotechnology applications.
Optical control of mammalian endogenous transcription and epigenetic states.
The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.