Search Results (4,183)

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 Popeye domain-containing protein 1 (Popdc1), also known as Bves, plays a crucial role in maintaining skeletal muscle homeostasis, with its variants leading to limb–girdle muscular dystrophy type R25. Skeletal muscles of patients with the homozygous missense variant of Bves exhibit impaired membrane trafficking, while skeletal muscle fibers in bves^S191F homozygous mutant zebrafish are significantly reduced and disorganized. However, the mechanism by which the absence of bves induces skeletal muscle atrophy remains unclear. In this study, we discovered a novel mechanism whereby bves deficiency drives skeletal muscle atrophy by disrupting mitochondrial structure and function. Our findings indicate that bves knockout leads to a significant decrease in zebrafish’s ability to swim, atrophy of skeletal muscle tissue, loss of cell membrane localization signals, and abnormalities in mitochondrial structure and function. After an 8-week intervention of regular aerobic exercise, the symptoms of skeletal muscle atrophy in bves knockout zebrafish were significantly alleviated, and the expression levels of genes and proteins related to mitochondrial were effectively rescued. These findings establish a connection between bves deficiency-induced disruption of mitochondrial structure and function and the onset and progression of skeletal muscle tissue atrophy symptoms, thereby laying a molecular foundation for exercise rehabilitation strategies in atrophic myopathy. 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

Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology. 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

Low-temperature-induced fertility restoration in thermo-sensitive genic male sterile (TGMS) lines severely impairs hybrid seed purity, which is a major bottleneck for two-line hybrid rice production. Most commercial TGMS lines rely on the single tms5 locus, leading to high climatic vulnerability. In this study, we developed a dual-locus strategy by target genome editing of TMS5 and MS1 in indica rice GH89. Adenine base editing at the MS1 locus exhibited a high editing efficiency of 93.5%. Transgene-free homozygous single mutants (GH89-tms5 and GH89-MS1) and double mutant (GH89-tms5 + MS1) were generated for phenotypic analysis. The double mutant GH89-tms5 + MS1 remained completely sterile for 5 and 10 days under controlled low temperature (23.5 °C), with only minimal fertility restoration after 15 days. In the field, it maintained complete sterility for 84 consecutive days and was fully insensitive to short-term low temperature fluctuations, outperforming single mutants and commercial control Y58S. Moreover, the double mutant retained most key yield-related agronomic traits of the wild type with only minor variations. This dual mutation forms a “double-lock” fertility regulatory system, significantly increasing the low-temperature duration threshold for fertility restoration. The GH89-tms5 + MS1 line exhibits promising potential for future rice breeding applications. Full article

Reparative macrophage polarization and macrophage-derived reactive oxygen species (ROS) are required for ischemia-induced revascularization in peripheral artery disease (PAD). Our previous study showed that mitochondrial fission protein dynamin-related protein 1 (DRP1) promotes reparative polarization and metabolic reprogramming in macrophages and post-ischemic neovascularization. However, the redox-dependent mechanism governing DRP1 activation in this context remains elusive. Here, using a mouse hindlimb ischemia (HLI) model of PAD, we identify cysteine sulfenylation (CysOH) of DRP1 as a critical redox modification induced in ischemic bone marrow (BM)-derived cells. BM chimeric mice reconstituted with CRISPR/Cas9-generated “redox-dead” DRP1-C631A knock-in mutant (Drp1^C/A) BM exhibited markedly reduced limb perfusion recovery and CD31⁺ capillary density in ischemic muscles following HLI. These defects were associated with enhanced Ly6G⁺ neutrophil accumulation, pro-inflammatory F4/80⁺CD80⁺ M1-like macrophages and reduced anti-inflammatory F4/80⁺CD206⁺ M2-like macrophages in ischemic muscle. Mechanistically, using an in vitro PAD model, hypoxia serum starvation (HSS) rapidly induced NADPH oxidase 2-dependent cytosolic ROS production and DRP1-CysOH formation in wild-type macrophages. In contrast, Drp1^C/A macrophages failed to undergo DRP1-CysOH-dependent mitochondrial fission under HSS, resulting in aberrant metabolic reprogramming characterized by enhanced glycolysis and mitochondrial ROS, pro-inflammatory p-NF-κB and M1-genes, and suppressed anti-inflammatory p-AMPK, efferocytosis and M2-genes. Thus, our findings establish DRP1 sulfenylation as a previously unrecognized redox-sensing mechanism that links ischemia-induced ROS to reparative macrophage reprogramming and revascularization, identifying a novel therapeutic target for PAD. Full article

Extreme climatic conditions often intensify abiotic stress factors (such as drought, salinity, heat stress, ultraviolet radiation, and soil degradation), and are increasingly limiting crop productivity and threatening global food security. Secondary metabolites (SMs), traditionally viewed as defense compounds, are now recognized as key regulators of plant adaptation to environmental stress. This review synthesizes recent advances in understanding the role of SMs as biochemical targets for improving crop resilience to climate extremes. By integrating evidence from multi-omics studies, artificial-intelligence-driven analyses, and functional genomics, we examine how stress-specific metabolic signatures and regulatory networks can be exploited for crop improvement. We further discuss the application of genome editing, synthetic biology, and metabolomics-assisted breeding to modulate the SM pathways to enhance stress tolerance. Selected case studies highlight the contribution of flavonoids, alkaloids, and terpenoids to stress adaptation in major and underutilized crops grown under salinity, drought, and low-temperature conditions. Despite significant progress, challenges remain, including metabolic trade-offs between stress tolerance and yield, regulatory constraints, and public acceptance of genetically engineered crops. By linking molecular mechanisms with applied strategies, this review provides a conceptual framework for leveraging secondary metabolism in climate-resilient agriculture and identifies key gaps to guide future research and innovation. 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

Bacterial spot, caused by Xanthomonas species, is a destructive tomato disease that reduces yield and fruit quality worldwide. The tomato resistance gene Rx4 confers hypersensitive response and field resistance to race T3 of Xanthomonas euvesicatoria pv. perforans, but downstream components associated with Rx4-mediated field resistance remain unclear. Here, we compared the susceptible processing tomato line OH 88119 with its near-isogenic line Rx4-1806 after spray inoculation with the race T3 strain Xv829. Transcriptome profiling at 1, 6, and 72 h post-inoculation identified limited transcriptional differences at 1 and 6 h, but 2247 differentially expressed genes at 72 h, including 1712 genes downregulated in Rx4-1806. Enrichment analyses highlighted plant–pathogen interaction, plant hormone signal transduction, and MAPK signaling pathways. Among candidate defense-related genes, SlWRKY33 was downregulated in Rx4-1806 and selected for functional validation. CRISPR/Cas9-mediated knockout of SlWRKY33 enhanced resistance in both OH 88119 and Rx4-1806, whereas SlWRKY33 overexpression increased infected leaf area and bacterial population. Knockout of SlPUB23 enhanced resistance in Rx4-1806 but not in OH 88119. These results suggest that SlWRKY33 and SlPUB23 negatively regulate tomato field resistance to bacterial spot race T3, with SlPUB23 functioning in an Rx4-dependent manner. 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