Search Results (391)

The innate immune system, particularly the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway, is a major early defense barrier against influenza A virus infection. However, excessive immune responses can trigger lethal cytokine storms and severe immune-mediated pathology. In this study, we performed a genome-wide CRISPR/dCas9 gene activation screen in human lung epithelial (A549) cells by using an A/Puerto Rico/8/1934 (H1N1) reporter virus, and identified the ubiquitin-specific protease USP17L13 as a novel negative regulator of innate immunity that promotes influenza virus replication. Overexpression of USP17L13 significantly enhanced the replication of multiple subtypes of influenza viruses in A549 cells, including a human pandemic H1N1 virus, seasonal H3N2 viruses, as well as a globally circulating clade, 2.3.4.4b, of the highly pathogenic avian H5N1 virus. Transcriptomic analysis demonstrated that USP17L13 suppresses host antiviral defenses by downregulating nuclear factor kappa B (NF-κB) signaling and arachidonic acid metabolism, while upregulating pathways associated with ribosomal translation and oxidative phosphorylation to facilitate viral production. Mechanistically, USP17L13 attenuates the host interferon (IFN) response by promoting the degradation of the key viral RNA sensors, RIG-I, and melanoma differentiation-associated protein 5 (MDA5). Further analysis revealed that USP17L13 is inducible by type I and type II interferons as well as inflammatory cytokines, suggesting that it may act as a negative-feedback regulator to limit excessive inflammation. Collectively, our findings identify USP17L13 as a previously unrecognized proviral host factor and provide new insight into how host deubiquitinases shape influenza virus-host interactions, with potential implications for host-directed approaches to controlling excessive inflammation during viral infection and improving influenza vaccine production. Full article

Protein turnover is essential for cellular metabolism, organelle biogenesis, stress adaptation, and ultimately the viability of cells and tissues. Papain-like cysteine proteases (PLCPs) are one of the vital components in protein degradation. PLCPs have been reported to act in senescence-associated proteolysis, but their roles in vegetative growth remain unclear. We identified PtCP1, an AALP-like PLCP in Populus tomentosa, localized to the vacuole and acid-triggered activated. CRISPR/Cas9-generated loss-of-function mutant (d7) showed dwarfism and non-stomatal photosynthetic limitations. On the other hand, the gain-of-function line (EM, deleted ERFNIN domain) exhibited accelerated growth and enhanced photosynthetic parameters. We showed d7 had the accumulation of Rubisco, which was the most important protein in photosynthetic carbon fixation. Transcriptomics revealed dysregulated carbon metabolism in d7. This data supported PtCP1-mediated proteolysis regulated photosynthetic carbon assimilation via altered Rubisco turnover, and then it increased the biomass accumulation during vegetative growth in woody plants. Full article

Tomato brown rugose fruit virus (ToBRFV) poses a major threat to global tomato (Solanum lycopersicum) production, as it can overcome conventional resistance genes that are effective against tobamoviruses. In this study, a multiplex CRISPR/Cas9 system was developed to target the SlTOM1 susceptibility gene family (SlTOM1a–d), which encodes host factors essential for tobamovirus replication. Six guide RNAs (gRNAs), designed following 12 off-target analyses, were assembled into a multiplex CRISPR/Cas9 construct using a Golden Gate cloning strategy and introduced into tomato genotypes through an Agrobacterium-based tissue culture transformation procedure. Although primary T₀ transformants exhibited chimeric mutation patterns, stable inheritance and segregation of edited alleles were confirmed in the T₁ generation. Sequence analyses identified diverse indel mutations across target loci, with SlTOM1d exhibiting the highest editing efficiency. Multiplex genome editing successfully generated single-, double-, and triple-mutant combinations, with higher-order mutants displaying the strongest tolerance phenotypes. Following mechanical ToBRFV inoculation, edited T₁ plants exhibited markedly reduced symptom severity, low viral accumulation, and improved fruit health compared to wild-type controls. RT-qPCR analysis further confirmed significantly reduced viral RNA levels, supporting a host-factor-mediated tolerance mechanism. Importantly, edited lines maintained normal growth and agronomic performance. Collectively, these findings demonstrate that multiplex CRISPR/Cas9-mediated targeting of SlTOM1 homologs represents a promising and practical strategy for improving ToBRFV tolerance in tomato breeding programs. Full article

The scarcity of nuclear male-sterile (NMS) lines severely constrains the development of hybrid breeding in soybean. This study highlights the important role of a conserved GmTGA9-GmDYT1 regulatory module in controlling soybean anther development and male fertility. Through CRISPR/Cas9-mediated screening of the four TGA9 genes in soybean, we discovered that GmTGA9c and GmTGA9d may be involved in male fertility. The tga9d single-gene mutation caused abnormal thickening of the anther middle layer and impeded anther dehiscence, resulting in partial male sterility. We then found that GmTGA9d directly bound to and inhibited its downstream target gene, GmDYT1c. Furthermore, genetic evidence supports that GmDYT1a and GmDYT1c have functionally overlapping roles. Mutation of both genes caused aberrant degradation of the anther middle layer and tapetum, interrupting microspore development and resulting in complete male sterility. This study provides evidence that the GmTGA9-GmDYT1 pathway in soybean plays important roles in regulating male sterility by controlling the development of the anther wall. These findings provide novel mechanistic insights for research on and application of NMS materials in soybean heterosis utilization and hybrid breeding. Full article

Phospholipase A2 group VI (PLA2G6) regulates phospholipid remodeling and cellular homeostasis, and its mutations cause neurodegenerative disorders, including neurodegeneration with brain iron accumulation and PLA2G6-associated parkinsonism (PARK14). Although many cases present in adulthood, a substantial subset shows early onset, indicating that PLA2G6 dysfunction can affect neuronal systems during developmental stages. However, whether PLA2G6 directly regulates early neurogenesis remains undefined. Here, using zebrafish embryos, we investigated the role of Pla2g6 during neural development through loss- and gain-of-function approaches. pla2g6 is dynamically expressed during embryogenesis, with enrichment in the developing central nervous system during neurogenesis. CRISPR/Cas9-mediated Pla2g6 deficiency did not alter neural progenitor formation but significantly reduced neuronal precursors. Expression of the disease-associated PLA2G6 D331Y variant phenocopied this effect, confirming that the observed phenotype results from loss of Pla2g6 function. The reduction in neuronal precursors occurred without changes in proliferation but was accompanied by a marked increase in apoptosis, identifying neuronal precursor cell death as the primary mechanism. Under oxidative stress conditions, Pla2g6 overexpression reduced neuronal apoptosis, whereas Pla2g6 deficiency markedly enhanced reactive oxygen species -induced apoptosis. These findings establish Pla2g6 as a regulator of oxidative stress-associated apoptotic signaling during neurogenesis. Together, these results define Pla2g6 as a stage-specific determinant of neuronal precursor survival, linking lipid homeostasis and oxidative stress control to early neural development. This study establishes a developmental framework for PLA2G6-associated disorders and positions impaired neuronal precursor survival as a contributing mechanism underlying disease onset. Full article

Background/Objectives: Rapid identification of foodborne pathogens is of high practical significance because it enables prompt epidemiological response, timely patient management, and effective sanitary control of food products. In this study, we developed an integrated molecular platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology for rapid, sensitive, and specific detection of Salmonella enterica. Methods: Four virulence genes (sirA, stn, siiD, and pagN) were selected as targets to ensure reliable pathogen identification. Reaction conditions were optimized using the Moraxella bovoculi Cas12a (MbCas12a) nuclease. The study focused on integrating isothermal amplification with a custom-engineered hardware solution for visual fluorescence detection. Results: The developed method demonstrated sensitive and specific detection, with no cross-reactivity to non-target microorganisms. Optimization allowed for a substantially reduced assay time of approximately 30 min. As a result, a portable fluorescence visualization approach was developed, featuring a 3D-printed housing and an integrated ultraviolet light source for direct visual fluorescence detection. This allows rapid differentiation of samples without specialized laboratory equipment, making it suitable for field applications. Conclusions: The combination of isothermal amplification, MbCas12a-based detection, and the portable fluorescence visualization approach provides a versatile platform for rapid diagnostics and food safety monitoring. This approach has strong potential to improve public health outcomes and enhance the resilience of food supply chains by enabling accessible, field-deployable pathogen detection. Full article

Soil salinization is one of the major abiotic stresses that influences agricultural production and the environment. Auxin response factors (ARFs) are key components of the auxin signal transduction pathway, while their role in wheat salt stress responses remains unclear. In this study, we identified TaARF18 as a negative regulator of salt tolerance in wheat. The coding sequences of TaARF18-A, TaARF18-B, and TaARF18-D were 2106, 2088, and 2088 bp, respectively. TaARF18 is a hydrophilic protein featuring typical Auxin-resp and B3 DNA-binding domains and exhibits relatively high evolutionary conservation among Poaceae species. The expression of TaARF18 was upregulated under salt stress. TaARF18 predominantly accumulated in the nucleus. Silencing of TaARF18 via the BSMV-VIGS approach enhanced salt tolerance in wheat seedlings. In addition, haplotype analysis based on resequencing data from 355 wheat accessions identified 25, 31, and 16 haplotypes for TaARF18-A, TaARF18-B, and TaARF18-D, respectively. Fourteen wheat accessions carrying different haplotypes were evaluated under salt stress, and HapIII of TaARF18-A exhibited the highest level of salt tolerance, which can act as a strong selection locus in global wheat breeding. Our findings provide insight into the function of ARFs in salt stress responses and offer a potential target for CRISPR/Cas-mediated salt-tolerant wheat breeding programs. Full article

Wheat is a globally important food crop, and its yield is crucial for ensuring food security. Lunxuan 13 is an elite wheat variety developed by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. It has high yield potential and outstanding agronomic traits, such as excellent seed setting rate, plump kernels, and good lodging resistance. However, this variety is highly susceptible to pre-harvest sprouting (PHS) when exposed to rain during the maturation period, leading to premature grain germination on the spike, which causes yield losses and quality deterioration, severely restricting its popularization. This study focused on addressing the PHS susceptibility of Lunxuan 13 by employing CRISPR/Cas9 technology for the targeted knockout of the three homoeologous copies (A, B, and D subgenomes) of TaQsd1, a key gene regulating seed dormancy. A total of 41 transgenic plants were obtained, achieving a transformation efficiency of 52.6%, among which 27 plants exhibited edits at the target sites, resulting in an editing efficiency of 65.9%. Phenotypic analysis of homozygous T₂ edited lines revealed significant functional redundancy among the three TaQsd1 homoeologs: a significant extension of the seed dormancy period and a substantial increase in PHS resistance were achieved only when all three A, B, and D copies underwent loss-of-function mutation (aabbdd genotype). After-ripened seeds from these mutants showed normal germination ability, indicating enhanced dormancy rather than loss of germination capacity. Importantly, all of the edited lines exhibited no significant differences compared to the wild type in key agronomic traits such as plant height, spike length, and grains per spike, thus retaining the excellent characteristics of Lunxuan 13. This study successfully optimized Lunxuan 13 for significantly enhanced PHS resistance while retaining its superior agronomic traits. This work provides an effective approach for improving PHS resistance in white-grained wheat and removes a key barrier to the potential commercialization of this variety. Full article

Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020–2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption—from days to less than one hour—while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements. Full article

Glucose deprivation is a major metabolic stress that requires coordinated adaptive responses to maintain cellular homeostasis and survival, yet the role of tripartite motif-containing 24 (TRIM24) in this process remains unclear. To address this question, we generated CRISPR-Cas9-mediated TRIM24-knockout MCF-7 and HEK293 cell lines, performed targeted metabolomic profiling and aspartate assays, used 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), aminooxyacetic acid (AOA), aspartate supplementation, and glutamic-oxaloacetic transaminase 2 (GOT2) knockdown to probe AMPK signaling and aspartate metabolism, and examined starvation responses in constitutive Trim24 knockout mice on a C57BL/6 background. Loss of TRIM24 sensitized cells to glucose deprivation. Re-expression of TRIM24 partially restored cell viability under glucose deprivation in both MCF-7 and HEK293 cells. Under glucose-free conditions, TRIM24 deficiency was associated with impaired AMP-activated protein kinase (AMPK) pathway activation, increased intracellular aspartate accumulation, and altered ATP/AMP levels. Pharmacological reactivation of AMPK by AICAR improved the survival of TRIM24-deficient cells under glucose deprivation. Reducing intracellular aspartate by AOA treatment or GOT2 knockdown restored AMPK pathway activation and improved adaptation to glucose deprivation, whereas exogenous aspartate suppressed AMPK signaling and increased ATP/AMP levels. In vivo, starvation of Trim24-deficient mice was associated with reduced AMPK pathway activation and increased aspartate levels. Together, these findings support a model in which TRIM24 contributes to adaptation to glucose deprivation and in which abnormal aspartate accumulation contributes to impaired AMPK pathway activation in TRIM24-deficient cells. Full article

Kv11.1 (hERG1) channels, encoded by KCNH2, mediate the rapid delayed rectifier potassium current (I_Kr) crucial for cardiac repolarization. Disruptions, via mutations or antiarrhythmic drugs like dofetilide cause severe arrhythmogenic disorders, including Long QT Syndrome Type 2 (LQT2), Brugada Syndrome (BrS), and Torsades de Pointes (TdP). While Kv11.1’s role in channelopathies and drug-induced arrhythmias is established, understanding its complex regulation and therapeutic targeting remains a challenge. This review synthesizes the structural, functional, and regulatory aspects of Kv11.1 channels and their clinical implications. Recent studies using iPSC-derived cardiomyocytes highlight regulation by PI3K/Akt, PKC, and PKA signaling via phosphorylation (Ser283, Ser890) and interactions with proteins like 14-3-3. Beyond electrophysiology, Kv11.1 influences pathological hypertrophy and non-cardiac functions including insulin secretion. Pharmacological efforts focus on activators to shorten action potential duration and suppress TdP, and blockers with overdose risks. Mutation heterogeneity, exemplified by trafficking impairment (G785D) in LQT2 and gain-of-function (R397C) in BrS, complicates precision therapy. Clinically, systematic risk stratification using electrocardiographic parameters and genotype-specific approaches enables personalized management. Beta-blockers remain first-line therapy for LQTS2, while rigorous avoidance of QT-prolonging medications and electrolyte monitoring form the cornerstones of preventive care. Advancing Kv11.1-targeted therapies with approaches like CRISPR-Cas9 and pharmacological chaperones (e.g., lumacaftor) holds promise for personalized treatments, ultimately reducing arrhythmic events and sudden cardiac death. Full article

Tumor initiation and metastatic progression are driven by context-dependent genetic alterations that disrupt tumor suppressor pathways, metabolic homeostasis, and signaling networks. However, the initial drivers that transform normal cells into malignant ones and their context dependency remain elusive. To address this, we aimed to systematically identify and characterize these drivers across cancer types, species, and microenvironments. We constructed customized clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout (KO) libraries targeting high-frequency mutated and downregulated genes associated with liver hepatocellular carcinoma (LIHC) and breast carcinoma (BRCA) and conducted parallel functional screens in non-cancerous mouse and human fibroblast cell lines under two-dimensional (2D), three-dimensional (3D), and in vivo conditions. Strikingly, TP53 and NF1 emerged as pan-context drivers consistently enriched across immortalization, tumorigenesis, and metastasis in both LIHC and BRCA settings, while most other identified drivers were largely species-, tissue-, and microenvironment-specific with limited cross-model overlap. Despite this heterogeneity, all drivers converge on core pathways including epigenetic regulation, metabolic reprogramming, and growth factor signaling. Unlike prior studies on established cancer cells, this work defines the genetic barriers restricting the malignant transformation of primary normal cells, offering a new framework for early cancer evolution. Full article

AGL6 genes are critical floral regulators in diverse angiosperms, yet their roles in legumes remain poorly understood. This study aimed to characterize GmAGL6 genes in soybean (Glycine max [L.] Merr. cv. Williams 82). We identified four homologs (GmAGL6a–d) featuring conserved MADS-box and K-box domains that cluster within the AGL6 lineage. Tissue-specific expression profiling revealed significant transcript enrichment during flower bud differentiation and maturation. Using CRISPR/Cas9, we generated quadruple knockout lines to evaluate gene function. Phenotypic analysis showed that, unlike the homeotic transformations typical of AGL6 loss in monocots, Gmagl6 quadruple mutants retained a standard papilionaceous floral structure without keel petal aberrations. However, the mutants did not show significant changes in floral height or width, but exhibited a significantly increased floral height-to-width ratio and smaller mature seeds, while vegetative architecture and podding capacity remained unaffected. These results suggest that GmAGL6 genes in soybean may function primarily in the regulation of floral proportion and seed development rather than floral organ identity. This research provides insights into the evolution of specialized legume flowers and suggests candidate genes for seed size improvement. Full article

MicroRNA-122 (miR-122) is the most abundant hepatic microRNA and a key regulator of hepatocyte proliferation, metabolism and differentiation. Although widely studied in hepatocellular carcinoma, its role in liver regeneration remains unexplored. This study investigated how miR-122 deficiency modulates liver regeneration under physiological conditions and during chronic liver injury. A miR-122-deficient mouse model (miR-122^−/−) was generated using CRISPR/Cas9, and liver regeneration was assessed after two-thirds partial hepatectomy (PHx) in healthy and CCl₄-induced fibrotic livers. In healthy liver, miR-122 expression was transiently downregulated within 24 h after PHx, suggesting a physiological role in cell cycle entry. After PHx in non-fibrotic livers, miR-122^−/− mice showed increased basal proliferation and accelerated regeneration, associated with Cyclin D1 and RhoA overexpression, enhanced cytokinesis and a predominance of diploid hepatocytes. In contrast, miR-122 deficiency markedly exacerbated CCl₄-induced fibrosis, leading to cirrhosis-like architecture, impaired hepatocyte function, and severe metabolic dysregulation. Despite increased proliferation after PHx, fibrotic miR-122^−/− mice exhibited severely impaired regeneration and near-complete mortality. Proteomic analyses revealed metabolic failure, oxidative stress, and inflammatory activation, creating an unfavorable environment for tissue repair. In conclusion, miR-122 plays a dual role in liver regeneration. While its suppression enhances regeneration in healthy liver, loss of miR-122 under fibrotic conditions drives pathological repair, metabolic failure and lethality, highlighting its critical role in chronic liver disease. Full article

Glucosamine (GlcN) is an essential amino monosaccharide widely used in pharmaceuticals, nutraceuticals, and cosmetics. Microbial fermentation presents a sustainable alternative to its traditional chemical production. However, in Saccharomyces cerevisiae, competitive carbon flux towards ethanol significantly limits GlcN yields. In this study, an S. cerevisiae strain for GlcN biosynthesis was engineered by integrating heterologous GlmD (glucosamine-6-phosphate deaminase) and GlmP (glucosamine-6-phosphate phosphatase) genes. To redirect carbon flux, the pyruvate decarboxylase genes pdc1, pdc5, and pdc6 were sequentially knocked out using the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach, generating strains S. cerevisiaeGlmDP/pdc1Δ, GlmDP/pdc1Δpdc5Δ, and GlmDP/pdc1Δpdc5Δpdc6Δ. S. cerevisiae GlmDP/pdc1Δpdc5Δpdc6Δ achieved a GlcN titer of 2.20 ± 0.11 g/L, a 1.54-fold increase over the parental S. cerevisia GlmDP strain, while its ethanol yield decreased by 26%. This enhancement was achieved without significantly affecting cell growth or glucose consumption. Comparative transcriptomics between the triple-knockout and parental yeasts revealed 892 differentially expressed genes. Pathways related to glycolysis and ethanol formation were predominantly downregulated, whereas pathways potentially supporting GlcN synthesis were upregulated. The engineered strain demonstrated high genetic stability over 50 generations. Our findings demonstrate that disrupting ethanol formation is an effective strategy to enhance GlcN production in S. cerevisiae, providing valuable insights for carbon flux redistribution. Full article