Search Results (1,330)

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disease (NMD) caused by loss-of-function mutations in the DMD gene. RNA-based therapies, especially antisense oligonucleotides (ASO)-mediated exon skipping and adenosine deaminase acting on RNA (ADAR)-guided RNA editing, have emerged as complementary approaches that modulate pre-mRNA splicing or correct transcripts without altering genomic DNA. Current phosphorodiamidate morpholino oligomer (PMO) drugs targeting exons 51, 53, and 45 provide mutation-class-specific benefit. At the same time, next-generation delivery strategies (e.g., peptide-conjugated PMOs (PPMOs), antibody–oligonucleotide conjugates (AOC), and endosomal-escape vehicles) aim to improve skeletal, cardiac, and diaphragm exposure. In parallel, RNA editing strategies offer a route to correct select nonsense or missense variants at the base level and may, in principle, restore near-native dystrophin expression. Meaningful translation of these modalities requires predictive large-animal models. A genome-edited microminipig (MMP) bearing DMD exon-23 mutations faithfully recapitulates hallmark features of human DMD. That includes early locomotor deficits, elevated serum creatine kinase (CK) and cardiac troponin T, progressive myocardial fibrosis, and a decline in left-ventricular ejection fraction (LVEF), while maintaining a manageable lifespan of approximately 30 months suitable for long-term studies. In particular, the MMP model provides a practical platform for addressing the persistent challenge of efficient therapeutic delivery to the heart and diaphragm through longitudinal dosing, imaging, and biopsy. In this review, we synthesize clinical progress in exon skipping, outline the promise of RNA editing, and integrate recent insights from Duchenne muscular dystrophy model for microminipigs (DMD-MMPs) as an advanced surrogate for preclinical development and translational evaluation. Full article

Background: The integration of multi-omics data, such as genomics and transcriptomics, into artificial intelligence models has advanced precision medicine. However, their clinical applicability remains limited due to model complexity. We integrated DNA mutation, RNA expression, and A>I(G) RNA editing data to develop a predictive model for drug response in breast cancer. Methods: We analyzed 104 patients from the Breast Cancer Genome-Guided Therapy Study (ClinicalTrials.gov: NCT02022202). Clinical variables, gene expression, tumor and germline DNA variants, and RNA editing features were integrated into machine learning models to predict therapy response. Generalized linear models (GLM), random forest (RF), and support vector machines (SVM) were trained and evaluated across multiple random 70/30 train-test splits. Feature selection was performed exclusively within the training set using LASSO regularization. Model performance was assessed using the F1-score on independent test sets. The additive effect of RNA editing was evaluated using paired comparisons across identical train/test splits. Results: We characterized the cohort using clinical, mutational, transcriptomic, and RNA editing profiles in 69 non-responders and 35 responders. Across repeated splits, adding RNA editing frequently maintained or modestly improved predictive performance, particularly in expression-based models, with paired analyses showing a statistically significant increase in F1-score. Conclusions: RNA editing represents a complementary molecular layer that can enhance multi-omic models for therapy response prediction in breast cancer, supporting further investigation of epitranscriptomic features in precision oncology. Full article

RNA editing is a critical post-transcriptional modification that contributes to transcriptomic and proteomic diversity. The most common A-to-I (recognized as G) RNA editing enzymes are adenosine deaminases acting on RNA 1 and 2 (ADAR1 and ADAR2, respectively), which mediate alterations across all regions of mRNA molecules. However, a systematic cross-tissue view of RNA editing and its molecular correlates is still lacking. Here, we developed a rapid method for ADAR editing assessment based on 24 frequently edited positions in coding regions, which enables faster estimation of RNA editing levels than previous methods. We applied this metric to assess RNA editing in normal and cancerous lung and colorectal tissues. We analyzed RNA and whole exome sequencing profiles of experimental 172 colorectal and 144 lung cancer samples, and literature 646 colorectal and 1037 lung cancer samples. We also examined two types of control tissues: tumor-matched normal tissues (51 colorectal and 108 lung samples) and healthy tissues (6 colorectal and 7 lung samples). Overall ADAR-mediated RNA editing levels were ~2.9- and ~4.7-fold higher in healthy controls than in colorectal and lung cancers, respectively. In addition to their well-known association with immune cells, we identified positive correlations of ADAR editing with 740 molecular pathways including those responsible for extracellular matrix organization, RAS-MAPK axis and G2/M phase cell cycle arrest, and negative—with 139 pathways responsible for DNA repair, apoptosis, expression of transposable elements, and other factors. Full article

The small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play important roles in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of different preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined. We therefore carried out an RNA-seq analysis and found that genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, and these effects were confirmed at both the RNA and protein level by RT-qPCR and Western blotting, respectively. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce genotoxic stress or apoptosis. Finally, the use of CRISPR/Cas9 genome editing and siRNA-mediated disruption of REV-ERB gene expression showed that the loss of the REV-ERB proteins did not impact the effect of SR8278 on gene expression and cell proliferation. We conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and, thus, care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown, particularly in studies involving cell proliferation. Full article

Corydalis impatiens (Papaveraceae) is a traditional Tibetan medicinal plant (“Pa Xia Ga”) whose mitochondrial genome evolution remains unexplored, particularly in the context of high-altitude adaptation. This study presents the first complete mitochondrial genome sequence of an alpine Corydalis species to establish a comparative framework with the lowland congener C. pauciovulata for investigating environment-associated mitochondrial evolution. Using Illumina sequencing and reference-guided assembly, we characterized a 688,959 bp circular genome containing 74 genes, with GC content variations reflecting functional compartmentalization—elevated in structural RNA genes (tRNAs: 51.24%; rRNAs: 52.79%) versus protein-coding genes (44.19%). We identified 719 RNA editing sites concentrated in NADH dehydrogenase genes, suggesting post-transcriptional optimization of respiratory complex I under hypoxic conditions. The genome harbors 50 dispersed repeats (7.50%) and 67 SSRs with A-rich predominance, providing species-specific markers for authenticating “Pa Xia Ga” in Tibetan medicine quality control. Phylogenomic analysis confirms close affinity with C. pauciovulata while resolving intrageneral relationships within Ranunculales. These findings establish a dual-reference system for distinguishing conserved genus-level features from altitude-associated adaptations, enabling future comparative mitogenomics across the 465-species genus and supporting DNA-based medicinal plant identification. Full article

Aspergillus flavus infection and accumulation of carcinogenic aflatoxins are detrimental to maize (Zea mays) production and consumption. We investigated lncRNA–RBP interactions during maize–A. flavus crosstalk using transcriptomic profiling, structural analysis, molecular docking simulations, and machine learning approaches. Analysis of 18 RNA-seq datasets identified 2104 lncRNAs in maize, of which 461 were differentially expressed under A. flavus infection. Distinct lncRNAs were preferentially induced under infection (e.g., Zm00001eb303170) or normal germination (e.g., Zm00001eb144150, Zm00001eb406410). RNA secondary structure predictions indicated high structural heterogeneity and thermodynamic stability, consistent with dynamic regulatory potential. Docking simulations with six key RNA binding proteins (RBPs)—including branch point bridging protein (BPB), KH domain protein, and pentatricopeptide repeat (PPR) proteins—demonstrated strong lncRNA–protein binding, with the lncRNA1–BPB complex exhibiting the highest binding affinity. ML algorithms identified the crucial role of tryptophan in determining interactions, while lncRNA17-KH and lncRNA1-BP complexes were found to have the best interaction under normal germination and A. flavus infection, respectively. The lncRNA–miRNA–mRNA regulatory network highlighted lncRNAs functioning as decoys or precursors of stress-responsive miRNAs (e.g., zma-miR156, zma-miR164, zma-miR399). These interactions targeted transcriptional regulators, splicing factors, and metabolic enzymes implicated in stress tolerance, seed germination, and systemic acquired resistance. The maize lncRNAs are active regulatory molecules embedded in complex RBP and miRNA interaction networks that fine-tune gene expression during A. flavus infection. The study provides novel insights into lncRNA-mediated resistance mechanisms and offers potential molecular targets for breeding or gene editing to mitigate aflatoxin contamination. Full article

The cultivated chrysanthemum is the most important ornamental species in the genus Chrysanthemum. However, because it is predominantly hexaploid and additionally exhibits self-incompatibility, it harbors numerous functionally redundant genes and displays extremely high heterozygosity. As a result, its genomic architecture is highly complex, making it challenging to interpret data obtained from omics analyses such as RNA-seq. To provide a genetically tractable model, we previously developed Gojo-0, a self-compatible, pure line of the diploid wild species C. seticuspe. In this study, we established Gojo-1, an improved self-compatible pure line derived from Gojo-0 and its sibling lines, exhibiting enhanced viability and culture performance. Leveraging these traits, we performed CRISPR–Cas9 editing of the AGAMOUS orthologs and successfully isolated mutants with altered floral organ morphology, demonstrating the line’s suitability for functional genomics. Comparative genome analysis showed that, aside from chromosome 1, the Gojo-1 genome is highly similar to that of Gojo-0, whose complete sequence has been determined. Taken together, these features indicate that Gojo-1 will serve as a valuable resource for future omics-based studies and a broad range of additional research applications. Full article

Over 70 mutations in the transforming growth factor beta-induced (TGFBI) gene are associated with corneal dystrophies that impair vision. The R555W hotspot mutation is a major cause of granular corneal dystrophy type 1 (GCD1). Here, we evaluated the technical feasibility of CRISPR/Cas9-mediated editing of the R555W mutation in peripheral blood mononuclear cells (PBMCs) obtained from a patient with GCD1. Three single guide RNAs (sgRNA1–3) and matched single-stranded oligodeoxynucleotide donors (ssODN1–3) were designed and co-transfected into PBMCs. Transfected cells were enriched by flow cytometric sorting, with GFP-positive cells representing approximately 2–4% of the total electroporated population. Editing outcomes were initially screened using high-resolution melting (HRM) analysis, and the sgRNA3–ssODN3 combination identified as the most promising candidate was subsequently validated by next-generation sequencing (NGS). Sequencing revealed a homology-directed repair efficiency of 98.2% among GFP-positive sorted cells, demonstrating efficient and precise genome editing within the enriched population. Because PBMCs are not disease-relevant corneal epithelial cells and only genomic endpoints were assessed, the clinical applicability of this study is limited and the work should be considered a technical proof-of-concept. This framework supports optimization of CRISPR-based strategies prior to studies in biologically relevant corneal models. Full article

A/T(U) and G/C nucleobase pair formation in DNA and RNA is crucial to numerous fundamental biological processes, including replication, transcription, and translation. The specificity of A/T(U) and G/C base pairing is used for the recognition of complementary sequences in medical and biotechnological applications, such as PCR, nucleic acid drugs, and CRISPR–Cas9-based gene editing. It is essential to understand and predict fidelity of biological reactions, avoiding off-target binding, in order to improve the accuracy and efficacy of applications. In particular, recognition mechanisms of complementary bases or whole sequences must be understood in detail. Despite the prevailing view that Watson–Crick hydrogen bonding is a primary mechanism for complementary base recognition, several experiments have shown that DNA polymerase does not require hydrogen bonding to select complementary bases. Other factors, such as the shape and geometric fitting of the bases and the base stacking, also appear to be crucially involved in the selection. E.g., artificial bases lacking the ability to form hydrogen bonds can still be recognized by DNA polymerase solely based on base-pair geometry. However, hydrogen bonding also contributes importantly to recognition. The accuracy of selecting a complementary nucleobase or sequence varies depending on reactions, suggesting the co-existence of multiple selection mechanisms. This review provides an overview of biological processes and applications involving base pairing and discusses the molecular mechanism underlying complementary base recognition. Full article

Gene editing, particularly CRISPR/Cas technology, represents a promising approach for the treatment of rare genetic diseases, including inherited retinal dystrophies, for which effective therapies are largely unavailable. Despite extensive research investigating gene editing across a wide range of cell types, transient delivery of CRISPR/Cas components and efficient homology-directed repair (HDR) in differentiated cells remain challenging. In this study, we employed hiPSCs derived from patients with Stargardt disease or Best disease, carrying pathogenic variants in ABCA4 or BEST1, respectively, to explore gene editing in human models. CRISPR/Cas9 and Cas12 nucleases were delivered into hiPS-derived retinal pigment epithelium (RPE) and retinal organoids using lipoplexes and compared with electroporation. We evaluated transfection efficiency, sgRNA-mediated DNA cleavage, and HDR-based correction. Precise repair of the pathogenic BEST1 variant was successfully achieved in hiPS-derived RPE cells using both nucleases, with Cas12 yielding the highest efficiency, exceeding 10% of HDR correction. Edited RPE cells preserved normal morphology and expressed specific maturity markers. In contrast, retinal organoids exhibited moderate transfection efficiency but showed no detectable CRISPR/Cas-induced DNA cleavage, highlighting the need for further optimization of gene editing in more complex cellular tissues. This study demonstrates, for the first time, precise correction of a single-nucleotide mutation in patient-derived RPE using CRISPR/Cas9 and Cas12 delivered using lipoplexes. These findings underscore the therapeutic potential of CRISPR/Cas-based strategies for inherited retinal dystrophies and provide a proof of concept for future clinical approximations. Full article

Abiotic stresses such as drought, salinity, extreme temperatures, and heavy metal contamination severely limit global crop productivity and threaten food security. Plants have evolved epigenetic strategies, particularly DNA methylation, to perceive, adapt to, and memorize environmental challenges. This review systematically elucidates the dynamic regulatory mechanisms of DNA methylation—including establishment via RNA-directed DNA methylation (RdDM), maintenance by methyltransferases (MET1, CMT), and active removal by demethylases (ROS1)—in plant responses to diverse abiotic stresses. We highlight how stress-induced methylation reprogramming modulates gene expression, chromatin states, and physiological adaptations, contributing to both somatic and transgenerational stress memory. Furthermore, we discuss advanced detection technologies for profiling methylation patterns and evaluate their applications in epigenetic breeding, such as exploiting heritable epialleles, RdDM-based gene silencing, and methylation markers for heterosis prediction. Despite significant progress, translating epigenetic insights into predictable breeding tools remains challenging. Future efforts should focus on establishing causal links between methylation changes and stress phenotypes, improving epigenome editing precision, and integrating multi-omics approaches for the development of climate-resilient crops. This work provides a comprehensive epigenetic perspective for enhancing crop adaptability and sustainable agriculture. Full article

Prime editing (PE) is a precise genome-editing technology that avoids double-strand breaks, holding great promise for clinical and agricultural applications. However, its genome-wide off-target effects are not fully understood, raising safety concerns. Here, we systematically compared the safety profiles of four prime editor variants (PE2max, PE3max, PE4max, and PE5max) using PEM-seq and RNA-seq. We further applied an ultra-sensitive method, Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI), to assess PE5max. Our results show that PE5max did not produce detectable sgRNA-dependent off-target single-nucleotide variants (SNVs) in the GOTI assay and induced only limited large deletions and chromosomal translocations. Collectively, this side-by-side benchmarking under matched conditions demonstrates that PE5max achieves an improved specificity profile, with no detectable increase in genome-wide off-target SNVs, advancing its potential for safer therapeutic use. Full article

Flemingia philippinensis Merr. et Rolfe (F. philippinensis) is a Chinese herbal medicine rich in polyphenols, especially isoflavone derivatives. It exhibits potent anti-inflammatory properties and is widely used in the treatment of various diseases. In this study, we aim to sequence, assemble, and analyze the mitogenome of F. philippinensis in detail to understand the genetic structure of their organelles and their gene expression. The results showed that the mitogenome of F. philippinensis possesses a circular architecture with a total length of 427,353 bp and a GC content of 44.90%. Annotation results revealed 33 unique protein-coding genes (PCGs), 16 transfer RNA (tRNA), and 3 ribosomal RNA (rRNA) genes in the mitogenome. Furthermore, comparative analysis of mitogenome andchloroplast gemone (cpgemone) sequences identified six mitochondrial plastid sequences (MTPTs), including one partial PCG and five complete tRNA genes. Subsequent collinearity analysis indicated that numerous homologous collinear blocks were detected between F. philippinensis and its closely related species, and have undergone a large number of genomic rearrangements in the F. philippinensis mitogenome. Finally, RNA editing analysis identified 498 C -to- U editing sites, notably enriched in nad4 (44 sites) and ccmB (33 sites). Codon usage bias analysis indicated that leucine (Leu 10.66%) and serine (Ser 9.19%) were the most frequently used amino acids. This study lays a theoretical foundation for further elucidating the structural characteristics and understanding the evolution, classification, and identification of F. philippinensis. Full article

Recent years have seen rapid progress in biological treatments for genetic diseases, as well as conditions like type 1 diabetes that lack an obvious genetic component. The authors sought to explain why this progress has emerged at this particular moment. The best way to illustrate this is by showcasing a wide range of therapies targeting diverse diseases. This progress has been driven by technological advances in genetically modified CAR-T and CAR-NK cells (e.g., using CRISPR or transgenes), which have led to significant improvements in cancer therapy. A key trend now is the emergence of “off-the-shelf” approaches aimed at generating cellular therapies compatible with a range of recipients by mitigating alloreactivity and immune rejection. Different diseases impose distinct biological and logistical limitations; thus, treatment of each patient requires an appropriate strategy. Emerging advances include the modification of therapeutic cells, either ex vivo or in vivo. Current options for transgene delivery mainly comprise lipid nanoparticles (LNPs), adeno-associated virus (AAV) vectors, and lentiviral vectors. Researchers also focus on selecting suitable promoters for specific expression in selected cell types. Altogether, these advances have led to remarkable progress in treating various diseases in recent years. This publication discusses the development of biological therapies, with particular emphasis on cell and gene therapies, illustrated by viable examples across various disorders. It covers implemented solutions for several types of cancer, as well as selected hereditary diseases and syndromes, including Huntington’s disease, carbamoyl phosphate synthetase 1 (CPS1) deficiency, hemiplegia, epidermolysis bullosa, chronic granulomatous disease, and congenital deafness. Emerging applications in heart diseases and diabetes are also summarized, along with therapeutic strategies involving tRNA gene editing. Although numerous strategies exist, only the most representative, practical, and up-to-date examples are emphasized. Full article

Extracellular vesicles, which carry bioactive cargos such as proteins, RNAs, and lipids, represent promising drug delivery vehicles owing to their biocompatibility, low immunogenicity, and inherent tissue-targeting capabilities. To address the current limitations in controlled cargo loading, we developed an abscisic acid (ABA)-inducible proximity system that directs proteins into exosomes during biogenesis. We engineered exosomal scaffolds by fusing the ABA receptor PYL1 to EV-enriched proteins—including BASP1, CD9, PTGFRN, and a truncated form PTGFRNΔ687—thereby creating docking sites within the exosomal lumen, while the target cargo (e.g., EGFP, firefly luciferase, or Cas9) was tagged with the ABI1 phosphatase domain. We demonstrate that ABA administration in producer cells induces PYL1–ABI1 complex formation, which recruits ABI1-fused cargo for selective encapsulation into EVs. Among the scaffolds tested, BASP1–PYL1 proved the most effective, enabling robust, ABA-dependent enrichment of cargo proteins. Purified EVs maintained canonical morphology, size, and marker expression (CD63, syntenin-1, CD9), confirming preserved biogenesis. Critically, these loaded exosomes efficiently delivered functional cargo to recipient cells, enabling Cas9/sgRNA-mediated genome editing. Together, our findings establish an ABA-triggered molecular switch for controllable EV protein loading, providing a versatile platform for next-generation therapeutic delivery. Full article