Search Results (1,359)

Background/Objectives: Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. Methods: We performed deep sequencing of ssODNs from three manufacturers and amplicons from edited hematopoietic stem/progenitor cells. Results: We find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional to their abundance in the ssODN. In our sickle cell mutation correction protocol, the most prevalent SNEs are predicted to produce benign β-globin variants, while the less frequent frameshift deletions are predicted to generate β-thalassemia-like alleles. Conclusions: Current quality control standards are insufficient to detect these errors, and deep sequencing of ssODNs should be incorporated into regulatory submissions for clinical gene editing programs. Full article

Chromosomal rearrangements that lead to the formation of oncogenic gene fusions, such as EML4-ALK, are thought to arise from incorrect repair of double-strand breaks in DNA. However, the mechanisms and factors driving rearrangement formation remain poorly understood, and analysis of these processes is limited by detection methods that are labor-intensive, low-throughput, and not readily quantitative at single-cell resolution. Here, we developed a genetically encoded ALK reporter based on A549 lung adenocarcinoma cells, created by inserting an ALK-P2A-mCherry cassette into the endogenous ALK locus, so that induced EML4-ALK fusion activated mCherry fluorescence. Reporter activation yielded a readily quantifiable mCherry-positive subpopulation that could be measured and enriched by flow cytometry and correlated with EML4-ALK levels. Using this platform, we combined CRISPR-mediated rearrangement induction with knockdown of DNA repair factors using RNA interference. Of the factors involved in base excision repair, homologous recombination-related pathways and canonical non-homologous end joining, knockdown of the APEX1 gene encoding apurinic endonuclease 1 (APE1) selectively increased EML4-ALK levels both in the reporter cell line and in parental A549 cells. Together, this work provides a sensitive, single-cell A549-based ALK reporter platform and a framework for future studies aimed at identifying cellular and environmental factors that modulate oncogenic EML4-ALK rearrangement formation. 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

Dairy cows are economically significant ruminants in China, and the dairy industry is closely linked to food safety and the agricultural economy. However, various factors such as pathogenic microorganisms often lead to frequent diseases in dairy cows. Furthermore, as potential hosts for diverse viruses, dairy cows can harbor zoonotic pathogens, which pose a threat to public health. The Shaanxi–Gansu–Ningxia region boasts abundant natural resources and extensive pastures. It is a major animal husbandry base in Northwest China, and dairy farming plays a significant role in the local economy. However, research on dairy cow virus diversity in this region remains limited; epidemic prevention and control capabilities are constrained, and the risk of disease outbreaks is elevated. In this study, 790 dairy cow samples were collected from 13 large-scale farms and free-range households in the Shaanxi–Gansu–Ningxia region from 2021 to 2023. Sample types consisted of nasal and anal swabs. Six viral metagenomic libraries were constructed and analyzed using high-throughput sequencing and bioinformatics methods, leading to the identification of 51 viral families. These comprised 16 positive-sense single-stranded RNA virus families, one Retroviridae family, four double-stranded RNA virus families, 21 double-stranded DNA virus families, and nine single-stranded DNA virus families. Among these, RNA viruses were represented by families such as Astroviridae, Coronaviridae, Caliciviridae, Picornaviridae, and Picobirnaviridae; DNA viruses were primarily detected in Circoviridae, Papillomaviridae, Genomoviridae, and Smacoviridae. Alpha diversity analysis revealed no significant differences in viral diversity and abundance among the three regions (p > 0.05); however, significant differences were observed in the read counts and proportions of RNA and DNA viruses across the provinces. Phylogenetic analysis further indicated that viruses carried by dairy cows exhibit considerable genetic diversity and pose potential cross-species transmission risks. This study established a reference database for the dairy cow virome in the Shaanxi–Gansu–Ningxia region, elucidated the phylogenetic relationships of key viruses, and provided a scientific basis for future monitoring and prevention of dairy cow viruses. Full article

Lactylation is an emerging lactate-derived post-translational modification that may link tumour metabolic reprogramming, epigenetic regulation and DNA damage repair. Enhanced glycolysis and lactate accumulation are common in many tumours, and lactate has been reported to induce histone and non-histone lactylation in specific experimental contexts. Recent studies suggest that lactylation is associated with several DNA repair pathways, including base excision repair/single-strand break repair, nucleotide excision repair, homologous recombination and non-homologous end joining, and may contribute to therapy resistance in selected cancer models. Specifically, XRCC1 lactylation has been reported to promote nuclear translocation and repair activity in glioblastoma models; H4K12 lactylation has been linked to PARP inhibitor resistance through RAD23A activation in ovarian cancer models; and BLM lactylation has been associated with enhanced homologous recombination repair in bladder cancer models. Lactylation of NBS1, RAD51 and XLF has also been implicated in DNA repair regulation in specific experimental systems, although some mechanistic links are inferred from pathway activation or functional rescue experiments rather than directly demonstrated across multiple tumour types. These findings suggest that lactylation may modulate DNA repair and therapeutic response in a context-dependent manner. Targeting lactate metabolism, transport and lactylation regulators, including LDHA, MCT1/4, ACAT1, AARS1 and GCN5, or using site-specific lactylation-inhibiting peptides may improve chemotherapy and PARP inhibitor efficacy, but clinical translation remains limited by heterogeneity, metabolic plasticity, toxicity and insufficient validation. 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

Reliable quality control of adeno-associated virus (AAV) vectors remains a major bottleneck in gene therapy manufacturing, particularly for resolving subtle differences in genome loading and conformation at the single-particle level. Existing approaches often struggle to distinguish AAV populations with similar mass and charge, such as capsids carrying self-complementary versus single-stranded DNA. Here, we introduce an AC plasmonic nanopore sensing framework for AAV9 characterization. Individual AAV capsids were optically trapped within a plasmonic double-nanohole nanopore and interrogated using multi-frequency AC pulse trains spanning 500 Hz to 100 kHz. To enhance sensitivity to localized particle–field interactions, a nanofabricated Ag/AgCl ring electrode was integrated concentrically with the plasmonic nanopore. Relative to a conventional wire electrode, the ring electrode produced broader and more robust analyte-dependent differences across multiple frequency-dependent parameters, enabling reliable discrimination of empty capsids (AAV_empty) and genome-loaded capsids carrying either self-complementary (AAV_scDNA) or single-stranded DNA (AAV_ssDNA), despite their near-identical genome mass. Concentration titration experiments further demonstrated that the extracted multivariate AC features remained largely concentration-independent over the tested range. Together, these results demonstrate that ring-electrode-enabled AC plasmonic nanopore sensing provides a multidimensional framework for resolving closely related AAV populations and advances plasmonic nanopores toward practical single-particle quality control of gene therapy vectors. Full article

Radiation-induced biological responses emerge through complex interactions across multiple biological scales, ranging from molecular damage to tissue remodeling and organism-level outcomes. Although traditional radiobiology has primarily focused on DNA damage and linear dose–response relationships, increasing evidence suggests that radiation responses are highly context-dependent and cannot be fully explained by genomic alterations alone. In particular, low-dose and chronic radiation exposures often induce biological effects that involve dynamic regulatory processes beyond direct mutational burden. The narrative review proposes a conceptual multiscale framework for predictive radiobiology that integrates genomic damage, post-transcriptional regulation, network rewiring, and tissue microenvironmental interactions. Within this framework, “predictive radiobiology” refers to the integrative prediction of radiation-induced outcomes, including radiosensitivity, tissue remodeling, fibrosis progression, therapeutic response, and long-term carcinogenic risk. We discuss how radiation-induced signaling extends beyond DNA double-strand breaks to include RNA-binding protein-mediated regulation, adaptive network responses, and extracellular matrix-dependent cellular plasticity. Recent advances in multi-omics, single-cell analysis, spatial biology, and three-dimensional organotypic models have revealed that radiation responses are governed by interconnected molecular and tissue-level processes. Furthermore, artificial intelligence and systems-level computational approaches provide new opportunities for modeling non-linear and context-dependent radiation effects across biological scales. We further discuss current limitations, including data integration challenges, reproducibility issues, and the translational gap between experimental models and clinical applications. Collectively, this conceptual framework highlights the need for integrative and multiscale approaches to improve mechanistic understanding and predictive modeling in modern radiobiology. Full article

Beyond its function as a carrier of hereditary information, recent research has uncovered novel properties of extracellular DNA, including its role in the adaptation to the environment when released from plants. The secreted DNA has been shown to exert insecticidal effects against insect pests, which play an adaptive role in plant-insect interactions, particularly in regulating populations of economically important sap-feeding insects. The molecular mechanisms underlying this insecticidal effect are underinvestigated and remain largely unknown. Therefore, there is a need for more efforts to uncover these mechanisms to better understand plant–pest interactions, which would provide new insights into natural pest control strategies and inspire biotechnological applications. In the current study, we show that Pittosporum tobira (P. tobira) secretes single-stranded DNA (ssDNA) that exerts an insecticidal effect on Coccus hesperidum (C. hesperidum). We collected extracellular DNA from P. tobira leaves and tested its potential insecticidal effect by applying it to C. hesperidum, which is a well-known pest that causes damage to P. tobira. Our results revealed that the outermost layer of the leaf cuticle of P. tobira predominantly contains ssDNA of approximately 100 nt in length, originating from both chloroplast and nuclear genomes. This DNA exhibited pronounced insecticidal activity against C. hesperidum, with chloroplast-derived sequences significantly enriched compared to the total DNA in intact plant cells. These findings suggest that the microevolution of the P. tobira nucleome and plastome contributed to the formation of extracellular DNA with insecticidal properties (eci-DNA), which is part of its defense strategy against insect pests. Moreover, in this article, for the first time, we show that antisense DNA (illustrated with oligonucleotide insecticide Coccus-11) is capable of activating insect retrotransposons and upregulating their RT-RNase H, a crucial enzyme for the DNA containment mechanism and successful action of oligonucleotide insecticides. Notably, the laboratory-developed ssDNA-based ‘genetic zipper’ technology, designed for sustainable pest management, possesses characteristics similar to eci-DNA found in nature, highlighting a potential natural parallel to this biotechnological approach for sustainable pest management. Full article

Duchenne muscular dystrophy (DMD) remains a major challenge in genetic medicine due to the difficulty of achieving durable, body-wide restoration of dystrophin in post-mitotic muscle tissues. Although current therapies—including exon skipping and micro-dystrophin gene replacement—have demonstrated clinical feasibility, their benefits are limited by incomplete efficacy, mutation specificity, and the need for repeated or high-dose interventions. These limitations highlight the need for strategies capable of directly and permanently correcting the underlying genetic defect. Recent advances in genome editing have positioned CRISPR-based technologies as promising candidates for this objective. Rather than functioning as a single approach, gene-editing platforms encompass a spectrum of strategies—including exon deletion, exon reframing, base editing, and prime editing—each with distinct advantages depending on the mutational context. In particular, the emergence of precision editing tools has enabled controlled nucleotide-level modifications, expanding the range of correctable mutations while reducing reliance on double-strand DNA breaks. In this review, we adopt a comparative and translational perspective to evaluate gene-editing strategies for DMD. We examine how different approaches align with specific mutation types, summarize key findings from preclinical studies, and analyze the major barriers to clinical implementation, including delivery efficiency, immune responses, editing durability, and genomic safety. We further discuss emerging innovations in editing technologies and delivery systems that aim to address these limitations. Collectively, this work reframes gene editing as a decision-oriented and application-driven therapeutic framework. Continued integration of advances in genome engineering, delivery platforms, and muscle biology will be essential to translate these technologies into safe, effective, and durable treatments capable of altering the clinical trajectory of DMD. Full article

Background/Objectives: The STAT (Signal Transducer and Activator of Transcription) family of seven transcription factors mediates cytokine and growth-factor signaling, regulating proliferation, differentiation, and immunity. While STAT3/STAT5 are established oncogenes and STAT1/STAT2 are classically viewed as tumor suppressors, emerging evidence indicates context-dependent roles in tumorigenesis. This study aimed to integrate evolutionary analysis with bulk transcriptomic, regulon, single-cell, and exploratory chromatin-binding analyses of the STAT family in human solid tumors. Methods: Orthologs and paralogs of human STAT genes (81 sequences total) were retrieved across vertebrates and invertebrates; a phylogenetic tree was constructed using MUSCLE alignment and Neighbor-Joining in MEGA12. Differential expression was assessed in TCGA solid tumors versus GTEx normal tissues. Master-regulator activity was inferred using the corto algorithm. Single-cell RNA-seq datasets were used to compare malignant and non-malignant cell populations. STAT1 chromatin binding was examined via ChIP-seq in interferon-stimulated HeLa and K562 cells. Results: Phylogeny resolved seven conserved vertebrate clades, with endocrine-responsive STAT3/STAT5 showing higher conservation and immune-associated STAT1/STAT2/STAT4/STAT6 exhibiting faster divergence. The majority of STAT genes were frequently upregulated across multiple solid tumors, with activated regulons confirming functional transcriptional engagement. Single-cell analysis demonstrated tumor-cell-autonomous upregulation of STAT1 and STAT2 in the HNSCC dataset. STAT1 ChIP-seq revealed asymmetric forward/reverse-strand read density around peak summits, supporting non-canonical DNA recognition. Conclusions: The STAT family operates as an evolutionarily conserved, broadly activated transcriptional module in human solid cancers, combining quantitative upregulation with qualitative shifts in DNA-binding dynamics. These findings refine our understanding of JAK/STAT signaling in oncology and highlight opportunities for network-targeted therapies. Full article

Enteroviruses are positive-sense single-stranded RNA viruses and common pathogens that are responsible for diverse public health diseases. To facilitate the study of the virus biology and pathogenesis of enterovirus, we developed a rapid method for construction of the enteroviral cDNA clones including enterovirus A71 (EV-A71) and coxsackievirus B5 (CVB5). As described for EV-A71, the full-length cDNA of CVB5 was amplified by long-distance PCR and cloned into a T7 promoter-containing plasmid using directional seamless cloning technology. The virus was successfully rescued by single transfection into cells stably expressing T7 polymerase and exhibited characteristics similar to the parental virus. Next, through systematic construction and the optimization of the EV-A71 and CVB5 reporter viruses, we successfully generated two novel reporter virus panels with high virus titers, rapid replication, and relatively stable genetic inheritance across passages using the new fluorescence proteins mScarlet3-H and the smallest miRFP670nano3. Analysis of critical determinants for the reporter virus construction revealed that reporter gene sizes, genomic insertion sites, and the usage of protease recognition sites are crucial parameters. The EV-A71 and CVB5 reporter viruses enable antiviral drug evaluation, as demonstrated by our identification of gemcitabine as a broad-spectrum inhibitor of both viruses. These systems also facilitate the functional interrogation of host factors, exemplified by our discovery that METTL3 promotes EV-A71 and CVB5 replication. These reverse genetic tools, including infectious cDNA clones and reporter viruses, will advance basic enterovirus biology and accelerate antiviral drug discovery. Full article

In biological systems, DNA serves as the primary carrier of genetic information, and the stability of its structure is fundamental to cellular function. Metal ions and temperature are critical environmental factors that modulate DNA conformation and activity. However, the differential morphological effects of alkali, alkaline earth, and transition metal ions, especially when combined with thermal treatment, have not been systematically visualized and quantified. In this work, atomic force microscopy (AFM) was employed to investigate the effects of different metal ions (Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺) and temperature on DNA structure. The results demonstrated that monovalent ions (Na⁺ and K⁺) neutralized the negative charges on the DNA backbone, thereby reducing intermolecular electrostatic repulsion and promoting DNA aggregation into dendritic structures. Divalent ions (Mg²⁺ and Ca²⁺) not only provided more effective charge screening but also formed ion bridges between DNA strands, leading to more compact and cross-linked networks. In contrast, Cu²⁺ ions directly coordinated with DNA bases, causing local structural distortion and strand scission. Elevated temperatures induced DNA melting, with distinct morphological transitions from extended double strands to condensed single-stranded globules observed at temperatures exceeding the melting point ($T_m$). These findings elucidate the mechanisms by which environmental factors govern DNA morphology, providing insights relevant to nanotechnology and molecular biology applications. Full article

Background/Objectives: TCIRG1-associated infantile osteopetrosis is a severe hereditary disorder caused by impaired osteoclast function, leading to osteosclerosis, hematological abnormalities, neurological complications, and early mortality. Early diagnosis and intervention are critical. Methods: A literature-based analysis was performed on clinical manifestations, outcomes of allogeneic hematopoietic stem cell transplantation (HSCT), immunomodulatory therapy, and experimental gene therapy and cell-based approaches, including lentiviral vectors and patient-derived induced pluripotent stem cells (iPSCs). Results: Allogeneic HSCT is the only established curative therapy, restoring osteoclast function and preventing severe complications. Early transplantation with HLA-matched donors and myeloablative conditioning provides optimal outcomes. Interferon γ1b can transiently enhance osteoclast activity but is not curative and shows variable efficacy. Preclinical studies demonstrate that lentiviral TCIRG1 delivery and transgenic correction in patient-derived iPSCs restore osteoclast function and bone resorption, with stable gene expression and minimal toxicity. Base and prime editing approaches offer potential for precise correction of single-nucleotide TCIRG1 variants, minimizing risks associated with double-strand DNA breaks. Conclusions: Allogeneic HSCT remains the standard therapy for TCIRG1-associated infantile osteopetrosis. Gene therapy and cell-based strategies represent promising adjuncts or alternatives, potentially avoiding immune-related complications and expanding therapeutic options. Further studies are needed to ensure safety, stable engraftment, and long-term efficacy, supporting translation of gene therapy into clinical practice. Full article

We examined neocortical pathology and interneuron degeneration in neonatal hypoxia-ischemic encephalopathy (HIE). Piglets in two age groups (2–3 or 7–10 days old, n = 4–12/group) underwent global cerebral hypoxia–ischemia (HI) or sham treatment. Piglets (2–3 days old) had epidural electrodes for continuous electroencephalography (cEEG) and were treated with hypothermia (HT) or remained at normothermia (NT). Older piglets, all NT, had scalp EEG. Piglets at both ages had seizures and survived for 1–7 days. Cortical damage was assessed by hematoxylin & eosin staining and immunohistochemistry; calretinin (CR), parvalbumin (PV), and vasoactive intestinal peptide (VIP) interneurons (INs) were counted. Cell injury was assessed by DNA fragmentation and protein nitration. TAR DNA binding protein-43 (TDP43) and the DNA repair scaffold protein X-ray repair cross complementing-1 (XRCC1) were examined for degeneration mechanisms. Cortical layers 3 and 4 showed high vulnerability; damage emerged as isolated cells, focal and laminar, and distributed as panlaminar throughout different cortical regions that correlated with seizure burden. HT protected strongly against cortical damage. CR- and PV-INs were severely depleted in HI-NT piglets compared to sham. VIP INs appeared invulnerable. HT partially rescued the loss of INs. CR and PV formed nuclear and cytoplasmic inclusions that colocalized with TDP43 and XRCC1; co-immunoprecipitation identified interactions among these proteins, and tyrosine nitration of CR. CR and PV INs accumulated DNA single- and double-strand breaks and appeared as attritional apoptosis variants with proteinopathy. This cell death is identified as aggreosis. IN loss correlated with seizure presence. Postmortem human neonatal HIE cases had a similar loss of CR and PV INs and nuclear depletion of TDP43 in the neocortex. Thus, neonatal HIE causes the loss of neocortical inhibitory IN subtypes with vulnerabilities instructed by their intrinsic calcium-binding protein signature and by mechanisms consistent with toxic sequestration and the nuclear depletion of XRCC1 and TDP43 underlying DNA damage accumulation. Early inhibitory IN deletion could drive seizure evolution in HIE; TDP43 and XRCC1 could be therapeutic targets for neonatal HIE. Full article