Search Results (379)

A new negative-strand RNA virus was identified in grapevines from a 38-year-old ‘Chardonnay’ block in Idaho through high-throughput sequencing (HTS) of total RNA. This virus was tentatively named grapevine-associated cogu-like Idaho virus (GaCLIdV). GaCLIdV has three negative-sense, single-stranded RNA genome segments of ca. 7 kb, 1.9 kb, and 1.3 kb, encoding L protein (RNA-dependent RNA polymerase, RdRP), a movement protein (MP), and a nucleocapsid protein (NC), respectively, identified based on pair-wise comparisons with other cogu- and cogu-like viruses. In phylogenetic analysis based on the RdRP, GaCLIdV grouped within the family Phenuiviridae and was placed in a lineage of plant-infecting phenuiviruses as a sister clade of the genus Laulavirus, clustering most closely with switchgrass phenui-like virus 1 (SgPLV-1) and more distantly related to grapevine-associated cogu-like viruses from the Laulavirus and Coguvirus clades. Both GaCLIdV and SgPhLV-1 are proposed to form a new genus, Switvirus, within the family Phenuiviridae. The presence of GaCLIdV in the original ‘Chardonnay’ samples was confirmed by RT-PCR amplification and Sanger sequencing. This new virus was found in five wine grape cultivars and in six vineyards sampled in Idaho and in Oregon during the 2020–2024 seasons. GaCLIdV may have contributed to the decline observed in the old ‘Chardonnay’ block, although the role of the virus in symptom development awaits further investigation. Full article

OCT4 is a critical transcription factor for early embryonic development and pluripotency. Previous studies have shown that the regulation of OCT4 by the transcription factor GATA4 is species-specific in pigs. This study aimed to further investigate whether there are other transcription factors that co-regulate the transcription of OCT4 with GATA4 in pigs. A CRISPR activation (CRISPRa) sgRNA library was designed and constructed, containing 5056 sgRNAs targeting the promoter region of 1264 transcription factors in pigs. Then, a pig PK15 cell line was engineered with a single-copy OCT4 promoter-driven EGFP reporter at the ROSA26 locus, combined with the dCas9-SAM system for transcriptional activation. The CRISPRa sgRNA lentiviral library was used to screen for transcription factors, with or without GATA4 overexpression. Flow cytometry combined with high-throughput sequencing identified MYC, SOX2, and PRDM14 as activators and OTX2 and CDX2 as repressors of OCT4. In the presence of GATA4, transcription factors such as SALL4 and STAT3 showed synergistic activation. Functional validation confirmed that HOXD13 upregulates OCT4, while OTX2 inhibits it. GATA4 and SALL4 synergistically enhance OCT4 expression. These findings provide new insights into combinatorial mechanisms that control the transcriptional regulation of OCT4 in pigs. Full article

Cool-season creeping bentgrass (Agrostis stolonifera L., As) is extensively used on golf courses worldwide and is negatively affected by several fungal diseases and abiotic stresses including drought and salinity. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) gene editing technology was employed in this project to knock out the AsDREBL (dehydration responsive element binding-like factor) gene, a potential negative regulator in stress tolerance. With our established single guide RNA (sgRNA)-based CRISPR-editing vector and optimized creeping bentgrass tissue culture system using mature seed-derived embryogenic calli of cv. Crenshaw as explant, more than 20 transgenic plants were produced by gene gun bombardment. Fifteen confirmed AsDREBL mutant plants were tested for drought and salinity tolerance by withholding water and applying salt spray in greenhouse settings. Some of the mutants were shown to be more tolerant of drought and salinity stress compared to the non-edited, wild type Crenshaw plants. Our results demonstrate that CRISPR-gene editing technology can be successfully applied to improve the agronomical traits of turfgrass. Full article

Galectin-3 is a multifunctional protein that is associated with diseases of the chorioretinal interface, in which the retinal pigment epithelium (RPE) plays a central role in disease development and progression. Since galectin-3 can function extracellularly as well as intracellularly via different mechanisms, we developed an immortalized human RPE cell line (ARPE-19) with a knockdown for galectin-3 expression (ARPE-19/LGALS3^+/−) using a sgRNA/Cas9 all-in-one expression vector. By Western blot analysis, a reduced galectin-3 expression of approximately 48 to 60% in heterozygous ARPE-19/LGALS3^+/− cells was observed when compared to native controls. Furthermore, ARPE-19/LGALS3^+/− cells displayed a flattened, elongated phenotype with decreased E-cadherin as well as enhanced N-cadherin and α-smooth muscle actin mRNA expression, indicating an epithelial–mesenchymal transition of the cells. Compared to wildtype controls, ARPE-19/LGALS3^+/− cells had significantly reduced metabolic activity to 86% and a substantially decreased proliferation to 73%. Furthermore, an enhanced cell adhesion and a diminished migration of immortalized galectin-3 knockdown RPE cells was observed compared to native ARPE-19 cells. Finally, by Western blot analysis, reduced pAKT, pERK1/2, and β-catenin signaling were detected in ARPE-19/LGALS3^+/− cells when compared to wildtype controls. In summary, in RPE cells, endogenous galectin-3 appears to be essential for maintaining the epithelial phenotype as well as cell biological functions such as metabolism, proliferation, or migration, effects that might be mediated via a decreased activity of the AKT, ERK1/2, and β-catenin signaling pathways. Full article

The CRISPR/Cas9 genome editing system has emerged as an effective platform to generate loss-of-function gene edits through non-homologous end joining (NHEJ) without a repair template. To verify whether small molecules can enhance the efficiency of CRISPR/ Cas9-mediated NHEJ gene editing in porcine cells, this experiment investigated the effects of six small-molecule compounds, namely Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, on the efficiency of CRISPR/Cas9-mediated NHEJ gene editing. The results showed the optimal concentrations of the small molecules, including Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, for in vitro-cultured PK15 viability. Compared with the control group, the single small molecules Repsox, Zidovudine, GSK-J4, and IOX1 increased the efficiency of NHEJ-mediated gene editing 3.16-fold, 1.17-fold, 1.16-fold, and 1.120-fold, respectively, in the Cas9-sgRNA RNP delivery system. There were no benefits when using YU238259 and GW843682X compared with the control group. In the CRISPR/Cas9 plasmid delivery system, the Repsox, Zidovudine, IOX1, and GSK-J4 treatments increased the efficiency of NHEJ-mediated gene editing 1.47-fold, 1.15-fold, 1.21-fold, and 1.23-fold, respectively, compared with the control group. Repsox can also improve the efficiency of NHEJ-mediated multi-gene editing based on a CRISPR sgRNA-tRNA array. We also explored the mechanism of Repsox’s effect on the efficiency of NHEJ-mediated gene editing. The results showed that Repsox reduces the expression levels of SMAD2, SMAD3, and SMAD4 in the TGF-β pathway, indicating that Repsox can increase the efficiency of CRISPR NHEJ-mediated gene editing in porcine cells through the TGF-β pathway. Full article

Rice seed storage proteins (SSPs) play a critical role in determining the nutritional quality, cooking properties, and digestibility of rice. To enhance seed quality, CRISPR/Cas9 genome editing was applied to modify SSP composition by targeting genes encoding 13 kDa prolamins and type A glutelins. Three CRISPR/Cas9 constructs were designed: one specific to the 13 kDa prolamin subfamily and two targeting conserved GluA glutelin regions. Edited T₀ and T₁ lines were generated and analyzed using InDel analysis, SDS-PAGE, Bradford assay, and RP-HPLC. Insertions were more frequent than deletions, accounting for 56% and 74% of mutations in prolamin and glutelin genes, respectively. Editing efficiency varied between sgRNAs. All lines with altered protein profiles contained InDels in target genes. SDS-PAGE confirmed the absence or reduction in bands corresponding to 13 kDa prolamins or GluA subunits, showing consistent profiles among lines carrying the same construct. Quantification revealed significant shifts in SSP composition, including increased albumin and globulin content. Prolamin-deficient lines showed reduced prolamins, while GluA-deficient lines exhibited increased prolamins. Total protein content was significantly elevated in all edited lines, suggesting enrichment in lysine-rich fractions. These findings demonstrate that CRISPR/Cas9-mediated editing of SSP genes can effectively reconfigure the rice protein profile and enhance its nutritional value. Full article

Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes. Full article

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering. Full article

In this study, we demonstrated that lrp6a, a co-receptor in the Wnt signaling pathway, is essential for proper median fin formation and somitogenesis in goldfish. We analyzed the gene’s sequence features and expression patterns in both wen-type and egg-type goldfish, uncovering distinct tissue-specific expression differences between the two varieties. To explore the functional role of lrp6a, we performed CRISPR/Cas9-mediated gene knockout using eight designed single-guide RNAs (sgRNAs), of which four showed effective targeting. Three high-efficiency sgRNAs were selected and co-injected into embryos to achieve complete gene disruption. Morphological assessments and X-ray microtomography (μCT) imaging of the resulting mutants revealed various abnormalities, including defects in the dorsal, caudal, and anal fins, as well as skeletal deformities near the caudal peduncle. These results confirm that lrp6a plays a key role in median fin development and axial patterning, offering new insights into the genetic regulation of fin formation in teleost fish. Full article

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder causing systemic skeletal dysplasia due to a deficiency of N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) enzyme activity, leading to the impaired degradation and accumulation of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate. While treatments such as enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available, they have significant limitations regarding efficacy in skeletal tissues and long-term safety, highlighting the need for more effective therapies. We evaluated a novel gene therapy approach using a dual Integrase-deficient lentiviral vector (IDLV) to deliver an expression cassette that includes human GALNS cDNA and Cas9 sgRNA, targeting the upstream region of the mouse Galns initial codon. This approach leverages the endogenous promoter to drive transgene expression. We assessed in vitro transduction, editing, and functional correction in NIH3T3 and MPS IVA mouse fibroblasts. In vivo efficacy was successfully evaluated via the facial vein injection in MPS IVA newborn mice. In vitro, this IDLV platform demonstrated supraphysiological GALNS activity in cell lysate, resulting in the normalization of KS levels. In vivo direct IDLV platform in newborn MPS IVA mice led to sustained plasma GALNS activity, reduced plasma KS, and favorable biodistribution. Partial correction of heart and bone pathology was observed, with no vector toxicity and minimal antibody responses. This dual IDLV-CRISPR/Cas9 approach effectively mediated targeted GALNS knock-in, yielding sustained enzyme activity, reduced KS storage, and partial pathological amelioration in MPS IVA mice. In conclusion, IDLVs represent an efficient, safe platform for delivering the CRISPR/Cas9 gene editing system for MPS IVA. Full article

Background/Objectives: Contextual memory associated with methamphetamine (METH) use contributes to relapse and persistence of addiction. Angiotensin II type 1 receptor (AT1R) signaling has been implicated in drug reinforcement. LCZ696, a clinically used combination of sacubitril (a neprilysin inhibitor) and valsartan (an AT1R antagonist), may interfere with METH-associated memory through the modulation of dopaminergic pathways. Methods: Male C57BL/6J mice were tested in a conditioned place preference (CPP) paradigm to assess the effects of LCZ696, sacubitril (AHU377), and valsartan on METH-induced memory expression and reinstatement. Synaptic plasticity in the nucleus accumbens (NAc) was examined by assessing the levels of synaptophysin (Syp) and postsynaptic density protein 95 (Psd95), as well as dendritic spine density. Dopaminergic signaling in the ventral tegmental area (VTA) was evaluated via ELISA, Western blotting, and chromatin immunoprecipitation (ChIP), targeting cAMP response element-binding protein (Creb) binding to the tyrosine hydroxylase (Th) promoter. To further assess the role of Th, an adeno-associated virus (AAV9) carrying a CRISPR-Cas9-based sgRNA targeting Th (AAV9-Th-sgRNA) was microinjected into the VTA. Results: LCZ696 and valsartan significantly reduced METH-induced CPP and reinstatement. LCZ696 reversed METH-induced synaptic and dopaminergic alterations and suppressed Creb-mediated Th transcription. Th knockdown attenuated both CPP acquisition and relapse. Conclusions: LCZ696 disrupts METH-associated contextual memory by modulating dopaminergic signaling and Creb-dependent Th expression, supporting its potential as a treatment for METH use disorder. Full article

Glaciers are significant sources of fresh water on planet Earth. The Hindukush–Karakoram–Himalayan (HKH) glaciers provide the water supply to more than half of the human population of the globe, for agricultural activities, biodiversity survival, and ecosystem services. In recent years, the loss of glacial ice has been forecasted to cause problems such as sea level rise, changes in water availability, and release of contaminants that reside in the surfaces of glaciers or within them. In this regard, mineralogical sediments play a significant role in the geochemistry of glaciers and element cycling. This study analyzed elemental pollutants found in the glaciers of Pakistan and investigated the diverse bacterial communities residing therein. Samples of ice and sediments were collected from the Gilgit, Hunza, and Swat glaciers in northern Pakistan. Nine elements, including co-factors, heavy metals, and nutrients, were assessed using atomic absorption spectrophotometry. The research findings indicate higher concentrations of the elements K, Fe, Cu, and Cr in Hunza glacier ice (Hgi) and Ni, Zn, As, and Cd in Gilgit glacier ice (Ggi). In terms of glacier sediments, Swat (Sgs), Gilgit (Ggs), and Hunza (Hgs) samples showed the highest concentrations of K, Cu, Ni, Zn, As, Pb, Cd, and, respectively, of Fe, and Cr. The amount of Cu and Cr is the same in Swat glacier ice and Swat glacier foot. However, the concentration of some elements (As, K, Pb, Zn) is higher in Swat glacier ice, while the amount of some elements (Cd, Ni) is greater in Swat glacier foot. Furthermore, microbial cultivation techniques revealed diverse bacterial communities inhabiting the sampled glaciers. Phylogenetic analysis of the bacterial isolates, based on 16S rRNA gene sequences, showed high homology (99–100%) with previously reported species. The resultant phylogenetic tree grouped the bacterial isolates, such as Serratia marcescens, Cupriavidus sp., and Bacillus cereus, with closely related species known for their roles in nutrient cycling, environmental resilience, and metal tolerance. These findings highlight the ecological significance and adaptive potential of microbial communities in glacier environments, emphasizing their role in elemental cycling and environmental resilience. Full article

Background: γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid with key roles in plant metabolism, stress responses, and fruit nutritional quality. In tomato (Solanum lycopersicum), GABA levels are dynamically regulated during fruit development but decline in the late ripening stages. Methods: To enhance GABA accumulation, we used CRISPR/Cas9 to edit the calmodulin-binding domain (CaMBD) of SlGAD2 and SlGAD3, which encode glutamate decarboxylases (GADs). The resulting truncated enzymes were expected to be constitutively active. We quantified GABA content in leaves and fruits and performed transcriptomic analysis on edited lines at the BR+7 fruit stage. Results: CaMBD truncation significantly increased GABA levels in both leaves and fruits. In gad2 sg1 lines, GABA levels increased by 3.5-fold in leaves and 3.2-fold in BR+10 fruits; in gad3 sg3 lines, increases of 2.8- and 2.5-fold were observed, respectively. RNA-seq analysis identified 1383 DEGs in gad2 #1−5 and 808 DEGs in gad3 #3−8, with 434 DEGs shared across both lines. These shared DEGs showed upregulation of GAD, GABA-T, and SSADH, and downregulation of stress-responsive transcription factors including WRKY46, ERF, and NAC. Notably, total free amino acid content and fruit morphology remained unchanged despite elevated GABA. Conclusions: CRISPR/Cas9-mediated editing of the CaMBD in SlGAD genes selectively enhances GABA biosynthesis in tomato without adverse effects on development or fruit quality. These lines offer a useful platform for GABA-centered metabolic engineering and provide insights into GABA’s role in transcriptional regulation during ripening. Full article

The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin–Darby bovine kidney (MDBK) cells is considerably limited. In this study, we have shown that siRNA-mediated depletion of TRIM5α, a restriction factor in HIV-1 infection, can dramatically enhance HIV-1 infection in MDBK cells. Furthermore, we generated a doxycycline-inducible Cas9-overexpressing MDBK cell line (MDBK-iCas9) suitable for CRISPR/Cas9-mediated editing. On this basis, we created a TRIM5α knock-out MDBK-iCas9 cell line MDBK-iCas9^{TRIM5α−/−} without additional genome insertions by combining sgRNA transfection and single-cell cloning. We found that MDBK-iCas9^{TRIM5α−/−} displayed greater permissiveness to lentivirus infection compared with MDBK-WT cells. Notably, we found that treatment with the chemical compound cyclosporine A, which directly interacts with cell factor cyclophilin A (CypA), could markedly increase the infectivity of lentivirus in both MDBK-iCas9^{TRIM5α−/−} and MDBK-WT cell lines, suggesting that CypA functions independently with TRIM5α as an inhibitor of the lentivirus in bovine cells. Therefore, combining bovine TRIM5α and CypA targeting could remarkably enhance lentivirus infection. In conclusion, our findings highlight a promising gene engineering strategy for bovine cells that can surmount the significant barriers to investigating the interplay between bovine viruses and their host cells. Full article

Stress granules (SG), dynamic cytoplasmic condensates formed via liquid-liquid phase separation (LLPS), serve as a critical hub for cellular stress adaptation and antiviral defense. By halting non-essential translation and sequestering viral RNA, SG restrict viral replication through multiple mechanisms, including PKR-eIF2α signaling, recruitment of antiviral proteins, and spatial isolation of viral components. However, viruses have evolved sophisticated strategies to subvert SG-mediated defenses, including proteolytic cleavage of SG nucleators, sequestration of core proteins into viral replication complexes, and modulation of stress-responsive pathways. This review highlights the dual roles of SG as both antiviral sentinels and targets of viral manipulation, emphasizing their interplay with innate immunity, autophagy, and apoptosis. Furthermore, viruses exploit SG heterogeneity and crosstalk with RNA granules like processing bodies (P-bodies, PB) to evade host defenses, while viral inclusion bodies (IBs) recruit SG components to create proviral microenvironments. Future research directions include elucidating spatiotemporal SG dynamics in vivo, dissecting compositional heterogeneity, and leveraging advanced technologies to unravel context-specific host-pathogen conflicts. This review about viruses and SG formation helps better understand the virus-host interaction and game process to develop new drug targets. Understanding these mechanisms not only advances virology but also informs innovative strategies to address immune escape mechanisms in viral infections. Full article