Search Results (1,771)

Abiotic stresses severely restrict plant growth, development, and crop yield. Histone modification functions as a key epigenetic regulator in plant stress adaptation. This review systematically summarizes the major types of histone modifications (e.g., acetylation, methylation) and their catalytic enzyme systems. It clarifies the regulatory patterns of chromatin remodeling and gene expression under diverse abiotic stress conditions, like extreme temperature changes, persistent drought, elevated salinity, and heavy metal exposure, and reveals the crosstalk networks between histone modifications and ABA, CBF/DREB, and ROS signaling pathways. It also discusses the transgenerational inheritance of stress-induced histone modification variations and their molecular basis, and introduces the application of CRISPR/Cas9 and dCas9-based epigenetic editing in improving crop stress resistance. Currently, research on histone modification in plateau crops remains fragmented: studies mostly focus on single stress rather than combined multiple abiotic stresses, lack tissue-specific epigenetic regulatory maps for native plateau plants, and the field application of epigenetic breeding technologies is seriously insufficient. Considering the compound stresses, including low temperature, drought, salinization, and heavy metals, on the Qinghai–Tibet Plateau, this review identifies current research gaps, such as tissue specificity, multi-stress crosstalk, and field application, and proposes future directions, including multi-omics analysis, stress adaptation mechanisms of plateau plants, and precise epigenetic breeding. Overall, this review fills the research gap of systematic collation on histone-mediated stress tolerance epigenetics under plateau combined abiotic stresses, and provides a theoretical reference for epigenetic research on plant stress resistance and for the improvement of plateau crops. Full article

TERT, the catalytic subunit of telomerase, is aberrantly activated in most cancers and represents an attractive therapeutic target. However, conventional TERT-targeting strategies, including chemical inhibitors and siRNA, are limited by several issues, such as insufficient efficacy and off-target effects. In this study, we investigated whether dCas9-KRAB-mediated CRISPR interference (CRISPRi) could overcome the limitations by transcriptional repression of TERT without DNA cleavage. We first assessed the efficacy of the dCas9-KRAB system by applying it to H1299 non-small-cell lung cancer cells and observed reduction in TERT expression up to approximately 80% and significant decreases in cell viability and growth. Transcriptome-wide analysis showed limited detectable changes in non-target-gene expression under the conditions tested. Together, the results suggest that dCas9-KRAB-mediated CRISPRi could serve as a proof-of-principle approach for targeted repression of TERT in cancer cells with limited detectable effects on non-target-gene expression. Full article

CRISPR-Cas9 has transformed microbial biotechnology by enabling precise genome modifications; however, achieving high editing efficiency remains a challenge due to multiple determinants, including on-target specificity, off-target events, PAM sequence, sgRNA scaffold composition, and RNA secondary structure. Our review foresees how artificial intelligence (AI) can address those challenges by enabling automated identification as well as highly active guide RNA (gRNA) optimisation. We highlight the influence of a data-driven training strategy that is focused on high-quality, diverse, and accurately labelled microbial datasets—mainly, given the limitations of models derived from mammalian systems that are not directly transferable to microbial organisms. Moreover, we discuss the key role of FAIR (Findable, Accessible, Interoperable, and Reusable) data principles and centralised, curated CRISPR-Cas databases as foundational elements for developing robust and predictive frameworks. Emerging directions are also explored, including generative AI approaches capable of supporting automated experimental planning. By considering the potential dual use of such technologies, the review further addresses bioethical considerations and regulatory frameworks necessary to ensure responsible genome engineering as a milestone, as well as the implementation of safeguards against misuse, particularly in pathogenic microorganisms. Furthermore, the convergence of standardised experimental data, specialised microbial datasets, and advanced AI architectures is paving the way to transform microbial biotechnology by accelerating metabolic engineering and synthetic biology applications. Full article

Variants in the human PAX4 gene are associated with both monogenic and complex forms of diabetes, yet their pathogenic effects remain difficult to define in models that accurately mimic human islet architecture and neonatal metabolic transitions. Here, we created a porcine PAX4 loss-of-function model using CRISPR/Cas9 cytidine deaminase base editing to introduce a premature stop codon in the PAX4 coding sequence. PAX4 knockout piglets developed severe hyperglycemia within 24 h of birth, followed by rapid postnatal clinical deterioration and uniform death by day 3. Biochemical analysis showed significant diabetic decompensation, including electrolyte imbalances, hyperosmolality, azotemia, dyslipidemia, and metabolic acidosis. Gross and histological examinations revealed notable pancreatic hypoplasia with preservation of exocrine tissue. Single-nucleus RNA sequencing and immunohistochemistry demonstrated an almost complete loss of insulin- and somatostatin-producing β- and δ-cells, respectively, with relative preservation of glucagon-expressing α-cells. Overall, these results establish PAX4 as a crucial factor in pancreatic endocrine development and postnatal glucose regulation in a large-animal model. This platform offers a human-relevant system for studying diabetes-associated PAX4 variants and for testing regenerative and gene-based therapies for insulin-deficient diabetes. Full article

Grapevine black-foot disease is a destructive trunk disease with a complex pathogen composition that often involves mixed and latent infections, making timely field diagnosis challenging. To improve rapid field detection, we developed a rapid, sensitive, and low instrument-dependent nucleic acid assay. The assay integrates recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a for the detection of Ilyonectria and Dactylonectria, two genera associated with grapevine black-foot disease. Conserved regions of the histone H3 and β-tubulin genes were selected for the design of specific RPA primers and corresponding CRISPR RNAs (crRNAs) for Ilyonectria and Dactylonectria, respectively. A workflow integrating RPA, Cas12a-mediated recognition, and lateral flow assay (LFA)-based visualization was established. The reaction conditions were optimized to enhance amplification efficiency and Cas12a recognition stability. Specificity was evaluated using DNA from target and non-target fungi, and sensitivity was determined using serially diluted templates. Under optimized conditions, the assay detected Ilyonectria DNA at concentrations as low as 3.6 ng/μL within 1 h at 39 °C. For Dactylonectria, the detection limit reached 80 fg/μL within 50 min at 41 °C. No cross-reactivity was observed. The LFA strips exhibited positive and negative bands within minutes, enabling rapid visual interpretation. This RPA-CRISPR/Cas12a-LFA system provides a rapid, visually interpretable approach for detecting selected grapevine black-foot disease-associated species in China. The workflow reduces the requirement for specialized thermocycling and fluorescence detection equipment during amplification and readout, following DNA extraction. Full article

Precise manipulation of mitochondrial DNA (mtDNA) by CRISPR-Cas systems remains challenging, largely due to inefficient import of guide RNAs, motivating the exploration of alternative programmable nucleases. Here, we show that prokaryotic Argonaute nucleases (pAgos) of various classes can be efficiently targeted to human mitochondria. Using the Su9 mitochondrial targeting sequence from Neurospora crassa, we achieved robust mitochondrial import of four pAgos—DecAgo, CbuAgo, KmaAgo and RslAgo. As a functional readout of their activity in cells, we targeted the single-stranded D-loop region, which plays a central role in mtDNA replication and maintenance, reasoning that cleavage at this site was expected to potentially result in a reduction in mtDNA copy number. Of the four enzymes, only RNA-guided DecAgo induced a pronounced reduction in mtDNA levels, decreasing copy number approximately fivefold within 48 h. Unexpectedly, this effect occurred independently of exogenous guides, suggesting that DecAgo may utilize endogenous mitochondrial guide RNAs. These findings identify DecAgo as an active nuclease in human mitochondria and reveal a previously unrecognized mode of targeting, highlighting the need to further investigate the underlying mechanism and the potential role of endogenous guide molecules, as well as improving targeting specificity. Full article

Early flowering-related factors play pivotal roles in coordinating plant growth and reproductive development. In this study, we investigated the biological function of early flowering gene (ELF) in Arabidopsis thaliana using CRISPR/Cas9-mediated genome editing and construction of overexpression approaches. Two independent ELF overexpression (OE-ELF) and genome-edited (ge-elf) lines were generated and systemically analyzed. ELF overexpression significantly enhanced early seedling performance, increasing germination rate and seedling fresh weight by up to 8.7%, while genome-edited lines exhibited a marked reduction. Root growth was strongly promoted in OE-ELF plants, with root length increase of 85% and 75%, whereas ge-elf lines showed a reduction of up to 48%. At later developmental stages, OE-ELF plants displayed enhanced vegetative growth, including increased leaf length (32%), leaf area (91%), and accelerated flowering (21% earlier than wild type). In contrast, ge-elf delayed flowering by up to 25% and resulted in compact plant architecture. Reproductive development was severely compromised in ge-elf plants, which exhibited malformed inflorescences, reduced pollen germination, shortened silique (45%), and a drastic decrease in seed number per silique (70%). Conversely, OE-ELF plants showed increased silique number and seed per silique. Molecular analysis revealed that ELF positively regulates key flowering-related genes, including FLC, SOC1, AP1, and LFY, which correlated strongly with growth and reproductive traits. Our results demonstrate that ELF functions as a central regulator integrating vegetative growth, floral development, male fertility, and seed production in Arabidopsis thaliana. Full article

The CRISPR/Cas9 system has been widely used for gene editing in various species; however, mosaicism remains a significant challenge. This study aimed to improve gene editing efficiency and reduce mosaicism in porcine embryos by exploring double electroporation pre- and post-in vitro fertilization combined with zona pellucida (ZP) removal. We evaluated the effects of these treatments on the development and mutation rates of oocytes/zygotes edited with guide RNAs (gRNAs) targeting GGTA1, CMAH, or B4GALNT2 genes. Double electroporation significantly increased the total and biallelic mutation rates in ZP-intact zygotes but not in ZP-free zygotes edited using GGTA1-targeted gRNAs. All blastocysts from ZP-free zygotes exhibited biallelic mutations following double electroporation. For the CMAH gene, all blastocysts exhibited mutations (biallelic mutations ≥ 80%); however, double electroporation and ZP removal did not affect their mutation rates or efficiency. For the B4GALNT2 gene, double electroporation significantly increased total mutation rates in ZP-intact zygotes, whereas all blastocysts from ZP-free zygotes showed biallelic mutation. These findings suggest that double electroporation, particularly with ZP removal, may enhance gene-editing efficiency, reduce mosaicism and improve the success of genetic modifications. Full article

Glutaric aciduria type 1 (GA1) is a rare neurometabolic disorder caused by glutaryl-CoA dehydrogenase (GCDH) deficiency, leading to the accumulation of neurotoxic metabolites that can cause both acute encephalopathic crises and progressive, insidious brain injury. Current management primarily relies on a protein-restricted diet, which remains therapeutically insufficient and burdensome for patients, highlighting the need for disease-modifying therapies. In this study, we established a novel GA1 mouse model using CRISPR/Cas9 technology and evaluated the preclinical efficacy of systemic recombinant adeno-associated virus (rAAV)-mediated gene therapy. Under standard dietary conditions without high-lysine challenge, our GA1 model exhibited sustained cerebral and hepatic glutaric acid (GA) accumulation and distinct chronic vacuolation in the hippocampus and cerebellum, mirroring the insidious-onset GA1 phenotype. Five-week-old mice received a single intravenous injection of rAAV-hGCDH using either rAAV2/8 or rAAV2/9 serotypes. Systemic rAAV-mediated gene therapy significantly reduced GA accumulation and attenuated chronic neuropathological changes in this GA1 mouse model for both serotypes. Our findings support the hypothesis that peripheral metabolic correction may play an important role in preventing the chronic neuropathological changes associated with GCDH deficiency. However, further investigation using tissue-specific expression systems is required to definitively delineate the relative contributions of hepatic versus central GCDH restoration to the observed neuroprotection. Full article

Background/Objectives: Immune checkpoints are critical regulatory pathways that maintain peripheral tolerance and prevent autoimmunity. Among these, the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) axis serves as a major inhibitory pathway that terminates T cell responses. While protein-based checkpoint blockade (ICB) targeting this axis has revolutionized clinical cancer therapy, its clinical efficacy is frequently limited by low response rates, immune-related adverse events (irAEs), and the emergence of adaptive resistance. To break through these bottlenecks, genetic interruption has emerged as a high-precision alternative to modulate the PD-1/PD-L1 pathway at the nucleotide level. Methods: A comprehensive systematic review of literature was performed across major databases (PubMed, Web of Science), with a focus on high quality studies published up to 2026. Results: Direct genomic disruption via CRISPR/Cas9 and post-transcriptional silencing through RNA interference can effectively neutralize inhibitory signaling at its source. Recent advances demonstrate that targeting upstream regulatory nodes—including metabolic checkpoints (e.g., lactate metabolism) and biophysical mechanisms (e.g., liquid–liquid phase separation)—provides superior transcriptional control over PD-L1. Furthermore, engineering CAR-T cells with multiplex gene editing (e.g., TCR/B2M/PD-1 knockout) or localized scFv secretion significantly enhances antitumor potency while reducing systemic toxicity. Innovations in organ-targeted lipid nanoparticles and stimuli-responsive biomimetic carriers further address the delivery barriers in solid tumors. Conclusions: Gene therapy provides a high-precision platform for PD-1/PD-L1 modulation, offering a viable strategy to overcome adaptive resistance. Future clinical application depends on the refinement of safer editing tools, such as base editing, and the standardization of intelligent delivery systems to ensure controllable and scalable cancer immunotherapy. Full article

The discovery of various CRISPR–Cas systems has revolutionized genome engineering by enabling precise and programmable nucleic acid targeting. Continued exploration of CRISPR diversity, together with advances in computational modeling and deep learning (DL)-based design, has expanded the potential to manipulate nearly any genomic locus, thereby accelerating both basic research and therapeutic applications. This review systematically provides a structured and up-to-date overview of CRISPR–Cas technologies, including their classification, computational modeling strategies, and the integration of machine learning (ML) and DL approaches to predict guide RNA (gRNA) efficiency and specificity. The emphasis is placed on studies published between 2019 and 2025, which highlight significant progress in modeling Cas–gRNA–DNA interactions, optimizing on/off-target prediction, and developing comprehensive CRISPR-related datasets. By synthesizing recent developments in CRISPR biology, computational simulations, and artificial intelligence, this review underscores the importance of interdisciplinary integration to improve the accuracy, safety, and scalability of next-generation genome-editing systems. Full article

CRISPR-based diagnostics now detect viral, bacterial, and cancer-associated nucleic acids with sensitivities approaching quantitative PCR; however, their translation to decentralized care rests on computational design and interpretation that current datasets cannot sustain. Pandemic-era Cas12a assays reached 95% positive predictive agreement against reverse transcription quantitative PCR (RT-qPCR) at 10 copies/μL, and deep neural networks now design Cas13 detection assays spanning 1933 vertebrate-infecting viruses, ranking candidate guides at Spearman correlations of 0.69 to 0.84 across internal and external validation. Generative deep-learning systems improve single-nucleotide discrimination two- to three-fold, computer vision classifies lateral flow outputs at 96.5% accuracy, and multi-biomarker fusion reaches an area under the receiver operating characteristic curve (AUC) of 0.998 in lung cancer detection. These results mask a narrow data foundation. Cas13a guide prediction still draws from a single screening library of 19,209 guide–target pairs, Cas12a has one published diagnostic model, and signal classifiers almost uniformly validate on single-site cohorts. This review synthesizes mechanistic constraints, predictive and generative models, and point-of-care classifiers, and maps the path beyond this data ceiling. Evolutionary pretraining on RNA corpora and lab-in-the-loop agents that convert model failure into targeted data acquisition define the route forward. Full article

Fish represent the most species-rich group within the phylum Chordata, possessing exceptional nutritional and ornamental value. Global aquaculture, particularly finfish farming, is experiencing rapid expansion worldwide, and fish serve as crucial model organisms for vertebrate developmental biology and functional genomics research. However, traditional breeding methods are plagued by limitations such as low precision and lengthy breeding cycles. Currently, gene editing technologies represented by the CRISPR/Cas system, base editing, and prime editing have provided revolutionary tools for dissecting gene function, modeling human diseases, targeted trait improvement, and ecological adaptation studies. This review describes the evolutionary history of gene editing technology, compares gene delivery strategies in fish embryos, and highlights landmark applications in key areas, including gene function research, aquaculture breeding, ornamental fish coloration regulation, and human disease model construction. Finally, we propose that innovation should be pursued while ensuring biosafety and regulatory compliance, to promote the transformation of fish gene editing toward large-scale and safe application. Full article

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

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