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So far, biocomputation strictly follows traditional design principles of digital electronics, which could reach their limits when assembling gene circuits of higher complexity. Here, by creating genetic variants of tristate buffers instead of using conventional logic gates as basic signal processing units, we introduce a tristate-based logic synthesis (TriLoS) framework for resource-efficient design of multi-layered gene networks capable of performing complex Boolean calculus within single-cell populations. This sets the stage for simple, modular, and low-interference mapping of various arithmetic logics of interest and an effectively enlarged engineering space within single cells. We not only construct computational gene networks running full adder and full subtractor operations at a cellular level but also describe a treatment paradigm building on programmable cell-based therapeutics, allowing for adjustable and disease-specific drug secretion logics in vivo. This work could foster the evolution of modern biocomputers to progress toward unexplored applications in precision medicine.
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Recent studies have suggested exosomes (EXO) as potential therapeutic tools for cardiovascular diseases, including atherosclerosis (AS). This study investigates the function of bone marrow stem cell (BMSC)-derived exosomes (EXO) on macrophage pyroptosis in AS and explores the associated mechanism. BMSC-EXO were isolated from healthy mice and identified. RAW264.7 cells (mouse macrophages) were exposed to oxLDL to simulate an AS condition. BMSC-EXO treatment enhanced viability and reduced lactate dehydrogenase release of macrophages. An animal model of AS was established using ApoE-/- mice. BMSC-EXO treatment suppressed plaque formation as well as macrophage and lipid infiltration in mouse aortic tissues. Moreover, BMSC-EXO decreased concentrations of pyroptosis-related markers interleukin (IL)-1ß, IL-18, cleaved-caspase-1 and gasdermin D in vitro and in vivo. Long non-coding RNA AU020206 was carried by the BMSC-EXO, and it bound to CCAAT enhancer binding protein beta (CEBPB) to block CEBPB-mediated transcriptional activation of NLR family pyrin domain containing 3 (NLRP3). Functional assays revealed that silencing of AU020206 aggravated macrophage pyroptosis and exacerbated AS symptoms in mice. These exacerbations were blocked upon CEBPB silencing but then restored after NLRP3 overexpression. In conclusion, this study demonstrates that AU020206 delivered by BMSC-EXO alleviates macrophage pyroptosis in AS by blocking CEBPB-mediated transcriptional activation of NLRP3.
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Aterosclerosis , Proteína beta Potenciadora de Unión a CCAAT , Exosomas , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Piroptosis , ARN Largo no Codificante , Animales , Masculino , Ratones , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Exosomas/genética , Exosomas/metabolismo , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Piroptosis/genética , Células RAW 264.7 , ARN Largo no Codificante/genéticaRESUMEN
Circumcoronene, a hexagonal graphene fragment with six zigzag edges, has been the focus of theoretical studies for many years, but its synthesis in solution has remained a challenge. In this study, we present a facile method for synthesizing three derivatives of circumcoronene using Brønsted/Lewis acid-mediated cyclization of vinyl ether or alkyne. Their structures were confirmed through X-ray crystallographic analysis. Bond length analysis, NMR measurement, and theoretical calculations showed that circumcoronene mostly follows Clar's bonding model and exhibits dominant local aromaticity. Its absorption and emission spectra are similar to those of the smaller hexagonal coronene due to its six-fold symmetry.
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INTRODUCTION: To devise a precise and efficient tool for predicting the individualized risk of acute-phase response (APR) in bisphosphonate (BP)-naive osteoporotic (OP) patients, receiving their first intravenous dose of zoledronate (ZOL). METHODS: The baseline clinical and laboratory data of 475 consecutive BP-naive OP patients, who received their first intravenous dose of ZOL between March 2016 and March 2021 in the Affiliated Kunshan Hospital of Jiangsu University, were chosen for analysis. Univariate and multivariable logistic regression models were generated to establish candidate predictors of APR fever risk, using three distinct fever thresholds, namely, 37.3 °C (model A), 38.0 °C (model B), and 38.5 °C (model C). Next, using predictor regression coefficients, three fever-threshold nomograms were developed. Discrimination, calibration, and clinical usefulness of each predicting models were then assessed using the area under the curve (AUC), calibration curve (CC), and decision curve analysis (DCA). The internal and external model validations were then performed. RESULTS: The stable predictors were age, serum 25-hydroxy vitamin D, serum total calcium, and peripheral blood erythrocytes count. These were negatively associated with the APR fever risk. The AUCs of models A, B, and C were 0.828 (95% confidence intervals [CI], 0.782 to 0.874), 0.825 (95% CI, 0.767 to 0.883), and 0.879 (95% CI, 0.824 to 0.934), respectively. Good agreement was observed between the predictions and observations in the CCs of all three nomograms. CONCLUSIONS: This study developed and validated nomogram prediction models that can predict APR fever risk in BP-naive OP patients receiving their first infusion of ZOL.
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Reacción de Fase Aguda , Difosfonatos , Reacción de Fase Aguda/inducido químicamente , Calcio , Difosfonatos/efectos adversos , Humanos , Estudios Retrospectivos , Vitamina D , Ácido ZoledrónicoRESUMEN
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.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Sistemas CRISPR-Cas , Diferenciación Celular , Reprogramación Celular , Epigénesis Genética , Luz , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células HEK293 , Células HeLa , Humanos , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Optogenética/métodos , Biología SintéticaRESUMEN
In this work, the waste biomass lotus leaf was converted into N-doped porous carbonaceous CO2 adsorbents. The synthesis process includes carbonization of lotus leaf, melamine post-treatment and KOH activation. For the resultant sorbents, high nitrogen content can be contained due to the melamine modification and advanced porous structure were formed by KOH etching. These samples were carefully characterized by different techniques and their CO2 adsorption properties were investigated in detail. These sorbents hold good CO2 adsorption abilities, up to 3.87 and 5.89 mmol/g at 25 and 0°C under 1 bar, respectively. By thorough investigation, the combined interplay of N content and narrow microporous volume was found to be responsible for the CO2 uptake for this series of sorbents. Together with the high CO2 adsorption abilities, these carbons also display excellent reversibility, high CO2/N2 selectivity, applicable heat of adsorption, fast CO2 adsorption kinetics and good dynamic CO2 adsorption capacity. This study reveals a universal method of obtaining N-doped porous carbonaceous sorbents from leaves. The low cost of raw materials accompanied by easy synthesis procedure disclose the enormous potential of leaves-based carbons in CO2 capture as well as many other applications.
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Lotus , Nitrógeno , Dióxido de Carbono , Hojas de la Planta , PorosidadRESUMEN
Synthesis of a carbon nanobelt (CNB) is a very challenging task in organic chemistry. Herein, we report the successful synthesis of an octabenzo[12]cyclacene based CNB (6), which can be regarded as a sidewall fragment of a (12,0) carbon nanotube. The key intermediate compound, a tetraepoxy nanobelt (5), was first synthesized by Diels-Alder reaction, and subsequent reductive aromatization gave the fully conjugated CNB 6. X-ray crystallographic analysis unambiguously confirmed the belt-shaped structure of 6. 1 Hâ NMR spectrum and theoretical calculations (ACID, NICS, and 2D/3D ICSS) revealed localized aromaticity and stronger shielding chemical environment in the inner region of the belt. The optical properties (absorption and emission) of 6 were studied and correlated to its electronic structure. Strain analysis indicates that the phenyl substituents at the zigzag edges are crucial to the successful synthesis of 6. This report presents a new strategy towards highly strained CNBs.
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Hepatogenous diabetes is a complex disease that is typified by the simultaneous presence of type 2 diabetes and many forms of liver disease. The chief pathogenic determinant in this pathophysiological network is insulin resistance (IR), an asymptomatic disease state in which impaired insulin signaling in target tissues initiates a variety of organ dysfunctions. However, pharmacotherapies targeting IR remain limited and are generally inapplicable for liver disease patients. Oleanolic acid (OA) is a plant-derived triterpenoid that is frequently used in Chinese medicine as a safe but slow-acting treatment in many liver disorders. Here, we utilized the congruent pharmacological activities of OA and glucagon-like-peptide 1 (GLP-1) in relieving IR and improving liver and pancreas functions and used a synthetic-biology-inspired design principle to engineer a therapeutic gene circuit that enables a concerted action of both drugs. In particular, OA-triggered short human GLP-1 (shGLP-1) expression in hepatogenous diabetic mice rapidly and simultaneously attenuated many disease-specific metabolic failures, whereas OA or shGLP-1 monotherapy failed to achieve corresponding therapeutic effects. Collectively, this work shows that rationally engineered synthetic gene circuits are capable of treating multifactorial diseases in a synergistic manner by multiplexing the targeting efficacies of single therapeutics.
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Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/terapia , Hepatopatías/complicaciones , Animales , Ingeniería Celular , Línea Celular , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Diseño de Fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Ingeniería Genética , Péptido 1 Similar al Glucagón/farmacología , Péptido 1 Similar al Glucagón/uso terapéutico , Humanos , Resistencia a la Insulina , Hepatopatías/metabolismo , Masculino , Ratones , Ratones Transgénicos , Ácido Oleanólico/farmacología , Ácido Oleanólico/uso terapéutico , Biología SintéticaRESUMEN
While federated learning (FL) is promising for efficient collaborative learning without revealing local data, it remains vulnerable to white-box privacy attacks, suffers from high communication overhead, and struggles to adapt to heterogeneous models. Federated distillation (FD) emerges as an alternative paradigm to tackle these challenges, which transfers knowledge among clients instead of model parameters. Nevertheless, challenges arise due to variations in local data distributions and the absence of a well-trained teacher model, which leads to misleading and ambiguous knowledge sharing that significantly degrades model performance. To address these issues, this paper proposes a selective knowledge sharing mechanism for FD, termed Selective-FD, to identify accurate and precise knowledge from local and ensemble predictions, respectively. Empirical studies, backed by theoretical insights, demonstrate that our approach enhances the generalization capabilities of the FD framework and consistently outperforms baseline methods. We anticipate our study to enable a privacy-preserving, communication-efficient, and heterogeneity-adaptive federated training framework.
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Given the severe protein denaturation and self-aggregation during the high-temperature desolubilization, denatured soy meal (DSM) is limited by its low reactivity, high viscosity, and poor water solubility. Preparing low-cost and high-performance adhesives with DSM as the key feedstock is still challenging. Herein, this study reveals a double-enzyme co-activation method targeting DSM with the glycosidic bonds in protein-carbohydrate complexes and partial amide bonds in protein, increasing the protein dispersion index from 10.2 % to 75.1 % improves the reactivity of DSM. The green crosslinker transglutaminase (TGase) constructs a robust adhesive isopeptide bond network with high water-resistant bonding strength comparable to chemical crosslinkers. The adhesive has demonstrated high dry/wet shear strength (2.56 and 0.93 MPa) for plywood. After molecular recombination by enzyme strategy, the adhesive had the proper viscosity, high reactivity, and strong water resistance. This research showcases a novel perspective on developing a DSM-based adhesive and blazes new avenues for changes in protein structural function and adhesive performance.
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Adhesivos , Glycine max , Transglutaminasas , Transglutaminasas/química , Transglutaminasas/metabolismo , Adhesivos/química , Glycine max/química , Glycine max/enzimología , Activación Enzimática , Viscosidad , Desnaturalización Proteica , Biomasa , Proteínas de Soja/químicaRESUMEN
The development of a biomass adhesive as a substitute for petroleum-derived adhesives has been considered a viable option. However, achieving both superior bonding strength and toughness in biomass adhesives remains a significant challenge. Inspired by the human skeletal muscles structure, this study reveals a promising supramolecular structure using tannin acid (TA) functionalized poly-ß-cyclodextrin (PCD) (TA@PCD) as elastic tissues and chitin nanocrystals (ChNCs) as green reinforcements to strengthen the soybean meal (SM) adhesive crosslinking network. TA@PCD acts as a dynamic crosslinker that facilitates reversible host-guest interactions, hydrogen bonds, and electrostatic interactions between adjacent stiff ChNCs and SM matrix, resulting in satisfactory strength and toughness. The resulting SM/TA@PCD/ChNCs-2 adhesive has demonstrated satisfactory wet and dry shear strength (1.25 MPa and 2.57 MPa, respectively), toughness (0.69 J), and long-term solvents resistance (80 d). Furthermore, the adhesive can exhibit desirable antimildew characteristics owing to the phenol hydroxyl groups of TA and amino groups of ChNCs. This work showcases an effective supramolecular chemistry strategy for fabricating high-performance biomass adhesives with great potential for practical applications.
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Quitina , Nanopartículas , Humanos , Nutrientes , Biomasa , Glycine max , Poli A , AdhesivosRESUMEN
Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A) signal of target genes and replacing it with a synthetic control region harboring RNA-binding protein (RBP)-specific aptamers, cap-dependent translation is rendered exclusively dependent on synthetic translation initiation factors (STIFs) containing different RBPs engineered to conditionally associate with different eIF4F-binding proteins (eIFBPs). This modular design framework facilitates the engineering of various gene switches and intracellular sensors responding to many user-defined trigger signals of interest, demonstrating tightly controlled, rapid and reversible regulation of transgene expression in mammalian cells as well as compatibility with various clinically applicable delivery routes of in vivo gene therapy. Therapeutic efficacy was demonstrated in two animal models. To exemplify disease treatments that require on-demand drug secretion, we show that a custom-designed gene switch triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice. For diseases that require instantaneous sense-and-response treatment programs, we create highly specific sensors for various subcellularly (mis)localized protein markers (such as cancer-related fusion proteins) and show that translation-based protein sensors can be used either alone or in combination with other cell-state classification strategies to create therapeutic biocomputers driving self-sufficient elimination of tumor cells in mice. This design strategy demonstrates unprecedented flexibility for translational regulation and could form the basis for a novel class of programmable gene therapies in vivo.
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Diabetes Mellitus Experimental , Animales , Ratones , Factor 4F Eucariótico de Iniciación/metabolismo , Procesamiento Proteico-Postraduccional , Regulación de la Expresión Génica , Proteínas Portadoras/metabolismo , MamíferosRESUMEN
Effects of hydrogen sulfide (H2S) on the browning and quality maintenance of fresh-cut peach fruit were studied. The results showed that H2S treatment repressed the development of surface browning, suppressed the increase in respiration rate and weight loss, and delayed the decline of firmness while soluble solids content (SSC) and microbial growth were unaffected during storage. H2S treatment maintained higher contents of phenolic compounds, especially neo-chlorogenic acid, catechin, and quercetin, and delayed the degradation of phenolic compounds by enhancing the activities of phenolic biosynthesis-related enzymes and inhibiting the oxidative activities of polyphenol oxidase (PPO) in comparison with control. Moreover, H2S stimulated the accumulation of amino acids and their derivatives including proline, γ-aminobutyric acid (GABA), and polyamines (PAs) via enhancing biosynthesis and repressing degradation compared to control. These results suggested that H2S treatment enhanced the accumulation of phenolic, amino acids, and their derivatives by modulating phenolic and amino acids metabolisms, which contributed to the higher antioxidant activity and membrane integrity maintenance, ultimately repressing browning development and maintaining the quality. Therefore, the current study speculated that H2S might be a promising approach for browning inhibition and quality maintenance in fresh-cut peach fruit.
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Exosomes (EXO) are extracellular vesicles with lipid bilayer membrane structure containing noncoding RNA, DNA, and other molecules which mediate biological functions. The importance of EXO derived from mesenchymal stem cells (MSCs) has been underlined in cardiovascular diseases. However, the functional role of long non-coding RNA (lncRNA) released by MSCs-EXO on atherosclerosis (AS) was unknown. We aimed to investigate the effects of lncRNA fetal-lethal non-coding developmental regulatory RNA (FENDRR) released from MSC-derived EXO on AS. The accumulation of oxidized low-density lipoprotein (oxLDL) caused AS in mice and damage to human vascular endothelial cells (HUV-EC-C). MSC-EXO restored HUV-EC-C activity and alleviated arterial injury. LncRNA microarrays revealed that FENDRR was delivered to cells and tissues by MSC-EXO. FENDRR bound to microRNA (miR)-28 to regulate TEA domain transcription factor 1 (TEAD1) expression. Moreover, FENDRR knockdown exacerbated cell injury and arterial injury in mice. miR-28 inhibitor reversed the effects of FENDRR silencing and reduced atherosclerotic plaque formation. While loss of TEAD1 mitigated the effect of miR-28 inhibitor and accentuated HUV-EC-C injury in vitro and AS symptoms in vivo. Our results demonstrated that MSC-EXO secreted FENDRR to treat AS. FENDRR competed with TEAD1 to bind to miR-28, thereby reducing HUV-EC-C injury and atherosclerotic plaque formation.
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Aterosclerosis , Exosomas , Células Madre Mesenquimatosas , MicroARNs , Placa Aterosclerótica , ARN Largo no Codificante , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Células Endoteliales/metabolismo , Exosomas/genética , Exosomas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados para ApoE , MicroARNs/genética , MicroARNs/metabolismo , Placa Aterosclerótica/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Effects of hydrogen sulfide (H2S) on chilling injury (CI), H2S, antioxidant and cell-wall metabolisms of refrigerated peaches treated with H2S and hypotaurine (HT, H2S scavenger) were investigated in present study. Results revealed that H2S treatment enhanced endogenous H2S content, which was associated with increased related H2S synthase enzymes activities, while HT showed the opposite results. Moreover, H2S treatment induced the accumulation of ascorbic acid, glutathione and the enhancement of antioxidant enzymes activities compared to control and HT, contributing to lower hydrogen peroxide content and superoxide radical production. Furthermore, H2S suppressed the increase of cell-wall degradation enzymes accompanied by higher levels of water-insoluble pectin, 24% KOH-soluble hemicellulose and cellulose, while HT accelerated these components degradation. Therefore, results indicated that H2S mitigated CI of refrigerated peaches by regulating H2S, antioxidant and cell-wall metabolisms, maintaining higher H2S and antioxidants contents, suppressing cell-wall degradation, thereby contributing to redox homeostasis maintenance and cell structure integrity.
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Sulfuro de Hidrógeno , Prunus persica , Antioxidantes/farmacología , Pared Celular/metabolismo , Frutas/metabolismo , Peróxido de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/química , Prunus persica/genética , Prunus persica/metabolismo , Plantones/metabolismoRESUMEN
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.
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Diabetes mellitus is a complex metabolic disease characterized by chronically deregulated blood-glucose levels. To restore glucose homeostasis, therapeutic strategies allowing well-controlled production and release of insulinogenic hormones into the blood circulation are required. In this chapter, we describe how mammalian cells can be engineered for applications in diabetes treatment. While closed-loop control systems provide automated and self-sufficient synchronization of glucose sensing and drug production, drug production in open-loop control systems is engineered to depend on exogenous user-defined trigger signals. Rational, robust, and reliable manufacture practices for mammalian cell engineering are essential for industrial-scale mass-production in view of clinical and commercial applications.
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Técnicas Biosensibles , Glucemia/metabolismo , Ingeniería Celular , Diabetes Mellitus Experimental/terapia , Terapia Genética , Péptido 1 Similar al Glucagón/genética , Células Secretoras de Insulina/trasplante , Insulina/genética , Biología Sintética , Animales , Biomarcadores/sangre , Encapsulación Celular , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Experimental/genética , Péptido 1 Similar al Glucagón/metabolismo , Células HEK293 , Homeostasis , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Ratones Endogámicos C57BL , TransfecciónRESUMEN
Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.
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Transferencia de Energía por Resonancia de Bioluminiscencia , Glucemia/metabolismo , Expresión Génica , Homeostasis , Optogenética , Transgenes , Animales , Línea Celular , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 1/sangre , Humanos , Cinética , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)-activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode-based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.
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Edición Génica , Neoplasias , Animales , Sistemas CRISPR-Cas/genética , Reparación del ADN por Unión de Extremidades , Humanos , Mamíferos/genética , Ratones , Neoplasias/genética , Neoplasias/terapia , OptogenéticaRESUMEN
Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel CaV1.2 and the inwardly rectifying potassium channel Kir2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human ß cells (Electroß cells). Wireless electrical stimulation of Electroß cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.