Search Results (4,169)

Global food production systems are increasingly challenged by population growth, climate change, water scarcity, and environmental degradation, necessitating the adoption of sustainable, resource-efficient food production strategies. Aquaponic systems integrate recirculating aquaculture with hydroponic crop cultivation, enabling nutrient recycling and improved water-use efficiency. Simultaneously, CRISPR/Cas9 genome-editing technology has emerged as a powerful tool for precise genetic improvement of economically important aquaculture traits. This review critically evaluates current progress in CRISPR/Cas9 applications in aquaculture, with emphasis on Nile tilapia (Oreochromis niloticus). Evidence from peer-reviewed studies indicates that targeted modification of genes associated with growth regulation, disease resistance, nutrient metabolism, feed efficiency, and stress tolerance can significantly enhance fish productivity and physiological resilience. Genes involved in hypoxia adaptation and nitrogen metabolism may further improve environmental performance in intensive recirculating systems by reducing ammonia accumulation and enhancing nutrient utilization. However, most genome-editing studies have been conducted under laboratory or conventional aquaculture conditions, with limited information available regarding the long-term performance, ecological interactions, microbial dynamics, and biosafety of genome-edited fish in aquaponic environments. Technical limitations including off-target effects, mosaicism, delivery efficiency, regulatory uncertainty, and public acceptance continue to constrain large-scale implementation. In the short term, CRISPR/Cas9 applications are likely to focus on practical trait enhancement under controlled aquaculture systems, whereas longer-term research may explore fish lines specifically optimized for nutrient cycling, environmental resilience, and integrated aquaponic sustainability. Overall, CRISPR/Cas9-mediated genome editing represents a promising but still emerging strategy for improving sustainable aquaculture and aquaponic food production systems. Full article

Glioblastoma (GBM) is an extremely invasive and incurable tumor. We previously reported predominant ALDH1A3 expression at the invasive front of GBM tumors, which was associated with shorter patient survival, and further showed that ALDH1A3 promoted tumor angiogenesis involving plasminogen activator inhibitor-1 (PAI-1). Here, we investigated whether ALDH1A3 drives cell invasion through retinoic acid (RA) and PAI-1 signaling. Analysis of the TCGA-GBM dataset revealed a positive association between ALDH1A3 and PAI-1 (SERPINE1) expression. Overexpression of ALDH1A3 in GBM cells markedly increased PAI-1 mRNA and protein levels, with cellular colocalization of both proteins, accompanied by robust migration and invasion. These effects were reversed by treatment with a pan-RA receptor (RAR) antagonist AGN193109 (AGN), with a specific PAI-1 inhibitor tiplaxtinin (Tip) or by CRISPR/Cas9-mediated knockout of PAI-1. In a chick chorioallantoic membrane (CAM) model, ALDH1A3-overexpressing cells showed increased invasion, which was reduced by tiplaxtinin (Tip) treatment or PAI-1 knockout. Mechanistically, ChIP-qPCR demonstrated that RA treatment or ALDH1A3 overexpression increased RARα occupancy at the PAI-1 regulatory region, accompanied by increased PAI-1 expression, both of which were diminished by AGN. Collectively, the present study defines an ALDH1A3-RA-PAI-1 signaling axis that contributes to GBM cell motility and invasion. Full article

Neospora caninum is a major cause of reproductive failure in cattle, responsible for epidemic abortion outbreaks that inflict annual billion-dollar losses on the global livestock industry. In water buffaloes (Bubalus bubalis), however, a phylogenetically close relative often raised in the same environments, the same parasite typically establishes a subclinical persistent infection with markedly lower rates of clinical abortion. This review inverts the traditional narrative by arguing that the key to next-generation control strategies lies in understanding the tolerant host (buffalo) rather than solely the susceptible host (cattle). By dissecting this “Neospora paradox”, we explore the molecular and immunological crosstalk that dictates pregnancy outcomes. We examine the parasite’s invasion proteins, revealed by CRISPR-Cas9 screens, and the maternal–fetal interface, where the balance between immune tolerance and parasite control determines the fate of pregnancy. We also compare N. caninum with the related zoonotic parasite Toxoplasma gondii to highlight how differential host immune recognition shapes infection outcomes. Finally, we propose that deciphering the buffalo’s successful equilibrium with N. caninum can illuminate novel pathways for vaccines and immunotherapeutic strategies, transforming the management of neosporosis worldwide. Full article

The Kelch-repeat superfamily genes played important roles in regulating plant growth and development; however, their functions in foxtail millet (Setaria italica) have not yet been characterized. In this study, SiNL4, a homolog of ZmNL4 controlling leaf width in maize, was knocked out using the CRISPR/Cas9 technology, and two homozygous knockout lines (ko1 and ko2) were obtained. Phenotypic analysis showed that compared with the wild-type Ci846, ko1 and ko2 exhibited reduced leaf width and decreased yield related traits (e.g., panicle weight, grain width, and 1000-grain weight). Cytological analysis showed that changes in leaf width of ko1 and ko2 resulted from a decrease in leaf epidermal cell width and the number of small vascular bundles (SVBs) close to the leaf edge, suggesting that SiNL4 might regulate leaf width by influencing cell expansion and the development of SVB. Spatiotemporal expression analysis indicated that the relative expression level of SiNL4 was high in the stem, leaf, and young panicle. Subcellular localization showed that SiNL4 was mainly localized in the mitochondria and plasma membrane. In addition, the T-DNA insertion mutant (Atnl4) of AT5G18590, the ortholog of SiNL4 in Arabidopsis thaliana, exhibited similar phenotypes with reduced rosette leaf width, seed width, and 1000-seed weight. Moreover, complementary expression of SiNL4 in Atnl4 not only restored the phenotypes, but also significantly increased the 1000-seed weight, indicating that the function of these two genes might be conserved. Meanwhile, we found that SiNL4 knockout caused a decrease in chlorophyll content and net photosynthetic rate (Pn), showing that SiNL4 might be involved in regulating photosynthesis. In summary, this study revealed the function of SiNL4 in regulating leaf width in foxtail millet, providing a potential gene for the genetic improvement of foxtail millet. Full article

Sickle Cell Disease (SCD) is a pervasive monogenic disorder characterized by chronic hemolytic anemia, unpredictable vaso-occlusive crises, and progressive multi-organ damage, representing a significant global health burden. Driven by a point mutation in the β-globin gene, the resulting abnormal Hemoglobin S (HbS) polymerizes under deoxygenated conditions, leading to erythrocyte sickling and systemic endothelial dysfunction. While supportive therapies such as hydroxyurea and transfusions manage symptoms, the mandate for definitive curative therapies is urgent. Historically, allogeneic hematopoietic stem cell transplantation (HSCT) utilizing matched sibling donors (MSD) has been the sole curative option, offering high survival rates but constrained by limited donor availability and the risk of graft-versus-host disease (GVHD). Consequently, alternative donor sources, including matched unrelated donors, umbilical cord blood, and haploidentical donors, have expanded patient access, particularly with the integration of post-transplant cyclophosphamide (PTCy) to mitigate alloreactivity. Concurrently, the advent of autologous gene therapy, encompassing lentiviral gene addition (Lyfgenia) and CRISPR-Cas9 gene editing (Casgevy) offers a revolutionary donor-independent approach that eliminates GVHD risk. Lyfgenia employs a lentiviral vector to introduce an anti-sickling βT87Q hemoglobin variant into autologous hematopoietic stem cells, while Casgevy employs CRISPR-Cas9 to disrupt the erythroid-specific enhancer of the BCL11A transcription factor, derepressing γ-globin expression and elevating fetal hemoglobin. This review synthesizes the pathophysiological mechanisms of SCD, evaluates the clinical outcomes and limitations of both allogeneic HSCT and autologous gene therapies, and outlines the clinical decision-making paradigms and future innovations required to achieve equitable global access to these transformative treatments. Full article

Forensic biotechnology is a rapidly evolving interdisciplinary field integrating molecular biology, genomics, and data science to address complex investigative challenges. Its applications span diverse domains, including criminalistics, food authentication, environmental monitoring, and bioterrorism preparedness. Advanced technologies such as Next-Generation Sequencing (NGS), CRISPR-Cas biosensors, and Artificial Intelligence (AI) play pivotal roles in modern diagnostics. NGS and eDNA revolutionize genetic profiling and ecological tracking, while microbiome analysis provides crucial insights into post-mortem intervals, cause of death, and geolocation. Simultaneously, CRISPR-based methods enable ultra-rapid pathogen detection, nanobiotechnology facilitates portable Lab-on-a-Chip (LOC) DNA analysis, and AI-driven algorithms optimize the interpretation of complex genomic mixtures and epigenetic age estimation. Despite these breakthroughs, significant challenges persist, including the strict legal admissibility of novel methodologies, the “black-box” dilemma in AI, ethical concerns regarding genetic privacy, and the critical need for global standardization. This review critically examines current biotechnological progress and future prospects, emphasizing the necessity of interdisciplinary collaboration to ensure reliable, accurate, and ethically sound forensic practices. Full article

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) is a major pathogen causing severe growth retardation in shrimp, leading to substantial economic losses in global aquaculture. To address the urgent need for accurate, rapid, and field-deployable diagnostic tools for EHP, this study developed a novel one-pot detection platform by integrating asymmetric Enzymatic Recombinase Amplification (aERA) with a PAM-independent CRISPR/Cas12a system (AYERA-Cas12a) based on ssDNA activation. This design circumvents the compatibility challenge between isothermal amplification and CRISPR activity in a single tube by generating single-stranded DNA amplicons that activate Cas12a without requiring a PAM sequence. The assay operates at a constant temperature of 46 °C and completes detection within 15 min. It achieves a sensitivity of 10 copies/μL, equivalent to qPCR, and shows no cross-reactivity with six other prevalent shrimp pathogens. Validation using 56 clinical shrimp (Litopenaeus vannamei, L. vannamei) samples demonstrated complete agreement with qPCR results. With its simple procedure, isothermal conditions, and clear endpoint fluorescence readout under blue light, the AYERA-Cas12a platform is suitable for point-of-care testing (POCT). This work provides a user-friendly tool for the on-site surveillance and early diagnosis of EHP, offering significant potential for improving disease management in shrimp farming. Full article

Plant architecture optimization is central to high-yield crop breeding. The number of branches in Brassica napus (B. napus) determines canopy structure, light use efficiency, and yield. The transcription factor BRANCHED1 (BRC1) integrates multiple signals to negatively regulate branching. This study characterized five BnaBRC1 homologs in B. napus via bioinformatics, expression profiling, and CRISPR/Cas9 editing. All BnaBRC1s contain a conserved TCP domain, and their promoters are enriched with light-responsive and hormone-responsive cis-acting elements. BnaA01.BRC1 is highly expressed in leaves, stem nodes, roots, and siliques, and its transcription is coordinately regulated by low light, sucrose, and exogenous cytokinin, and gibberellin (GA) signals. Functional analysis showed that overexpression of BnaA01.BRC1 suppressed branching, whereas CRISPR/Cas9-mediated knockout of BnaBRC1 substantially increased branch number. In basal axillary buds, high BnaBRC1 expression was accompanied by upregulation of GA-inactivating GIBBERELLIN 2 OXIDASEs and the GA signaling negative regulator SPINDLY, and no direct interaction was detected between BnaA01.BRC1 and DELLA proteins, suggesting indirect regulation of branching via GA homeostasis. Collectively, this study demonstrates the pivotal role of BnaA01.BRC1 in branching regulation and provides a genetic resource and theoretical basis for plant architecture optimization and multi-branch germplasm innovation in B. napus. Full article

Rapeseed (Brassica napus L.) growth and productivity are severely constrained by drought stress worldwide. Stomata are central regulators of plant transpiration and gas exchange, and therefore, represent key targets for enhancing water-use efficiency and drought tolerance. The transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), a key regulator of the abscisic acid (ABA) signaling pathway, plays crucial roles in plant abiotic stress responses and stomatal regulation. Nevertheless, the biological functions of BnaABI4 in B. napus remain largely unclear. In this study, four BnaABI4 paralogs were identified in the elite rapeseed cultivar ZS11 through genome-wide identification and comprehensive bioinformatic analyses. Each BnaABI4 protein harbors only one conserved AP2 domain, and their promoters contain multiple stress/hormone-responsive cis-regulatory elements (CREs). We subsequently generated BnaABI4-4 overexpression (OE) lines as well as BnaABI4 CRISPR/Cas9-mediated knockout (KO) transgenic lines. Phenotypic assays demonstrated that OE line had reduced transpiration rate (Tr), stomatal conductance (Gs) and stomatal density, along with enhanced drought tolerance, whereas KO lines showed the opposite phenotype. Transcriptome profiling identified numerous differentially expressed genes (DEGs) enriched in biological pathways associated with stomatal regulation, ABA signal transduction, and drought acclimation. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses confirmed significant enrichment of DEGs in processes including stomatal development, stomatal movement, reactive oxygen species (ROS) homeostasis, and drought tolerance. Collectively, our findings demonstrate that BnaABI4 negatively regulates stomatal density while positively contributing to drought tolerance in B. napus. This study lays a mechanistic foundation for genetic improvement and molecular breeding of drought-tolerant rapeseed cultivars. Full article

One of the major obstacles in early cancer detection in dogs is the limited sensitivity in detecting circulating tumor DNAs (ctDNAs) with low abundances. Standard next-generation sequencing (NGS) without error correction typically achieves detection limits around ~1% mutant allele frequency (MAF). We sought to improve the detection sensitivity using a sequential CRISPR-EspCas9 enrichment strategy in which iterative in vitro cleavage (IVC) was combined with PCR amplification to selectively deplete wild-type DNA and enrich rare tumor mutations. Applying the strategy to genomic DNA and cell-free DNA mimics from canine mammary gland tumor cell lines demonstrated that IVC enrichment enabled the detection of cancer-associated PIK3CA H1047R mutations that were undetectable by conventional Sanger sequencing. To evaluate detection sensitivity, we characterized enrichment using synthetic templates for PIK3CA H1047R and other cancer-related mutations, BRAF V596E, and KRAS G12C. We observed that three iterations of sequential IVC achieved ~160, ~15, and ~2.2-fold enrichment for PIK3CA H1047R, BRAF V596E, and KRAS G12C, respectively. Under the present synthetic-template conditions, the analytical LOD reached 0.001% MAF for PIK3CA and 0.01% MAF for BRAF, whereas KRAS showed only modest enrichment and remained practically limited under the current guide design. Together, the results show that the CRISPR-EspCas9 IVC strategy enables selective enrichment of low-frequency single-nucleotide mutant alleles. We anticipate that the finding could be utilized to develop a highly sensitive veterinary liquid biopsy application with further optimization and validation using canine plasma cfDNA. Full article

Background and Objectives: Among CNS malignancies arising in infancy, ATRT stands out as the most frequently diagnosed in children younger than six months. Disruption of the SMARCB1 gene underlies the overwhelming majority of cases. Progress toward effective treatment has been hampered by two persistent challenges. Current mouse models, while informative, fall short of reproducing the full clinical and biological picture of human ATRT, and their ability to predict therapeutic outcomes in patients remains uncertain. Compounding this, the rarity of the disease makes it difficult to assemble patient cohorts of sufficient size for meaningful clinical trials. At the molecular level, germline loss of SMARCB1 exons 4 and 5 has emerged as a particularly penetrant predisposing event, with affected individuals presenting at an earlier age than those harboring other mutation types. The porcine SMARCB1 gene offers a compelling basis for translational modeling as its protein product is identical to the human ortholog at every amino acid position across isoforms, a degree of conservation that exceeds what is seen in the mouse. Methods: Thus, we hypothesized that germline deletion of exons 4 and 5 would predispose heterozygote swine to ATRT development. In this manuscript, we describe the creation of an ATRT porcine model through a CRISPR/Cas9 mediated gene-editing approach. Results: 15 piglets were produced, two of which had confirmed SMARCB1 targeted excisions. However, none developed tumors. To induce further tumorigenicity, one pig with confirmed exons 4 and 5 excision was crossed with a pig with TP53 exon 2 truncation. In total, 11 piglets were born, of which one contained the original excision without a TP53 mutation. This piglet developed a spinal mass at the T1 level. Conclusion: To our knowledge, this is the first ATRT porcine model ever developed and provides proof-of-concept feasibility for large animal modeling of SMARCB1-deficient rhabdoid tumors. These findings support the continued development of porcine RTPS-1 models toward preclinical application. Full article

Bemisia tabaci (whitefly; Hemiptera: Aleyrodidae), a complex of morphologically similar but genetically distinct species, causes enormous agricultural damage worldwide. Farmers incur billions of dollars in losses each year from whiteflies, both through direct feeding damage and from the transmission of numerous plant viruses. Important crops that are heavily damaged by whiteflies include tomato, eggplant, cucumber, cotton, cucurbits, beans, and cassava. The global invasiveness and persistence of B. tabaci are largely attributed to its exceptional biological traits. Understanding these traits is essential for developing effective, long-term pest management strategies. This review describes in detail how the basic biology studies of B. tabaci provide a foundation for developing pest management strategies. Specifically, we discuss: (1) insights into the development of insecticide resistance can guide resistance management strategies; (2) knowledge of natural enemies supports the advancement of biological control approaches; and (3) understanding plant–insect interactions reveals molecular targets for innovative pest management solutions. We also examine emerging research trends and offer future perspectives on how ongoing studies may drive the development of next-generation control strategies (RNA interference, clustered regularly interspaced short palindromic repeats—CRISPR-associated protein 9 (CRISPR-Cas9), and horizontally transferred genes as targets). Full article

Ezrin, expressed by the EZR gene, is a member of the ERM protein family that connects the plasma membrane to the actin cytoskeleton, participating in processes such as cell adhesion, migration, and signaling. However, its role in cardiac morphogenesis remains incompletely understood. In zebrafish (Danio rerio), two ezrin homologs, ezra and ezrb, are present. CRISPR/Cas9 gene editing technology was used to generate ezra knockout lines, and the simultaneous knockdown of ezra and ezrb was induced via morpholino oligonucleotides (MOs). To investigate the molecular mechanisms, transcriptome sequencing and bioinformatic analysis were conducted on 48 h post-fertilization (hpf) ezrin–MO embryos, with subsequent validation using a real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH) experiment. The results showed that ezra^−/− exhibited a compensatory upregulation of ezrb without overt developmental defects, whereas ezrin–MO embryos presented with pericardial edema, reduced cardiac chamber size, and atrioventricular valve malformations at 48 hpf. RNA-seq revealed that myocardial contraction-related genes were significantly dysregulated and apoptotic signaling pathways were activated in ezrin–MO embryos. These findings demonstrate that ezra and ezrb are functionally redundant in cardiac development and that the loss of ezrin function may lead to cardiac developmental defects and impaired myocardial contractility via the activation of apoptotic signaling pathways. Full article

It is crucial to consider newer antibiotics with activity against anaerobes and Helicobacter pylori, given their healthcare importance, and the constantly growing antibiotic resistance/multidrug resistance, which complicates the therapy. The aim of this review was to emphasize certain recently approved or still-under-investigation antibiotics with potential benefits for treating Clostridioides difficile infections (CDIs), other anaerobic infections, and those caused by H. pylori, covering recent data from articles published primarily in 2020–2026. Given the limited number of antibiotics for treating CDI and fidaxomicin nonavailability in many countries, it is necessary to conduct more extensive laboratory and clinical studies of promising antibiotics such as ibezapolstat, delafloxacin, lascufloxacin, omadacycline, eravacycline, ridinilazole, and CRS3123. Against Bacteroides fragilis group species, delafloxacin and eravacycline showed good activity. Research on rifasutenizol for bacterial vaginosis, sarecycline and nadifloxacin for acne vulgaris and amixicile for periodontal diseases needs to be expanded. For H. pylori infection, delafloxacin, sitafloxacin, nemonoxacin, zoliflodacin, and rifasutenizol may improve the suboptimal success of most eradication regimens. However, more efforts, in coordination between medical, scientific, manufacturing, and government representatives, should ensure wider access to and research on the newer antibacterials. Establishing more research groups, careful examination of market issues, and additional approaches, such as nanomaterials, efflux pump inhibitors, phage therapy, and CRISPR-Cas systems, should be beneficial. Notwithstanding the difficulties, there are many opportunities to promote research on and potential use of newer antibiotics which show advantages over the older antibacterials, and to make them available to numerous countries and patients worldwide. Full article

Hulunbuir short-tailed sheep (HSTS) and Hu sheep (HS) exhibit distinct tail phenotypes linked to ecological adaptation, with HSTS carrying a loss-of-function mutation (c.G334T) in the T gene while HS retain the wild-type allele. However, the cellular and molecular mechanisms underlying T-mediated tail development remain unclear. Here, we performed single-cell RNA sequencing on HSTS and HS embryos at embryonic days 16 and 19 (E16 and E19), complemented by cross-species validation using a CRISPR/Cas9 mouse model carrying the same mutation. We identified 12 cell types in E16 HSTS and E16 HS embryos, and 15 cell types in E19 HSTS and E19 HS embryos and found that the MDK_ITGA6+ITGB1 ligand–receptor pair consistently mediated core intercellular communication. The MDK_ITGA6+ITGB1 axis mediates intercellular communication critical for tail bud formation; BMP activation and FGF repression disrupt AER survival, leading to tail shortening. Developmental trajectories showed a shift from early progenitor states at E16 to terminal differentiation at E19. Crucially, HSTS embryos showed transcriptomic signatures consistent with premature AER regression. The T mutation showed transcriptomic signatures of increased BMP pathway activity and reduced FGF8 expression, which may disrupt AER survival and contribute to the short-tail phenotype. In the mouse model, mutant T expression was reduced, and expression dynamics of WNT5B and FGF8 were perturbed, corroborating the sheep findings; however, homozygous T mutation causes embryonic lethality in mice but not in sheep, indicating species-specific differences. This study provides single-cell transcriptomic evidence linking the T c.G334T mutation to premature AER regression in sheep, complemented by cross-species validation in a CRISPR/Cas9 mouse model, offering new insights into the cellular mechanisms of tail development and may provide a basis for future investigations into tail-related breeding markers, pending experimental validation. These changes are associated with AER maintenance and tail outgrowth. Full article