Search Results (2,678)

The plant mitochondrial genome has become a current research hotspot as an independent genetic model. Nevertheless, mitochondrial genome information for most Dendrobium species remains unknown. In this study, the assembly of mitochondrial genome of Dendrobium nobile Lindl.,1830 and Dendrobium denneanum Kerr., 1933 was conducted through the application of second- and third-generation sequencing technologies, with the mitochondrial genome of D. denneanum Kerr. being reported first. The results revealed that the mitochondrial genomes of the two species possessed a multi-chromosome circular structure. Their total lengths were 641,414 bp and 558,760 bp, consisting of 21 and 19 contigs, respectively. A total of 67 and 72 genes, 993 and 1491 repeat sequences, and 549 and 553 RNA editing sites were identified. Gene loss was observed. A total of 26 and 36 homologous fragments were detected between the mitochondrial and the chloroplast genome, accounting for 5.09% and 4.93% of the total lengths, respectively, indicating intracellular gene transfer. Synteny and phylogenetic analyses revealed that the two species shared extensive collinear regions and clustered together in a distinct clade of the phylogenetic tree, indicating a close sister relationship. These findings enrich the mitochondrial genome database and provide valuable insights to guide future research on species identification and molecular evolution of the genus Dendrobium. Full article

Allogeneic hematopoietic cell transplantation (HCT) has been used for decades to treat certain malignant and non-malignant hematological conditions, but challenges remain. Increased understanding of disease mechanisms and recent developments in genome editing have enabled alternative strategies utilizing gene-edited autologous HCT and many of these have progressed to the clinic. We present here a comprehensive review of clinical trials of gene-edited autologous hematopoietic stem cells for the treatment of hemoglobinopathies and immunodeficiencies. Searches of major international clinical trial registries were carried out using specific key words. In total, 44 interventional clinical trials investigating gene-edited autologous stem cell therapies were identified, with CASGEVY (exagamglogene autotemcel) being the only product approved to date. Hemoglobinopathies were the most common indication (n = 37) followed by immunodeficiencies (n = 4), with single trials in HIV-1 infection, pyruvate kinase deficiency and limb–girdle muscular dystrophy. Gene-editing strategies fall into three categories: disruption of the BCL11A erythroid enhancer, editing of the γ-globin promoter and direct correction or disruption of disease-relevant genes. CD34⁺ hematopoietic stem and progenitor cells are the most common cell types edited, and CRISPR-Cas9 is the most widely used gene-editing modality. While results are encouraging, efficient intracellular delivery of gene-editing tools, editing efficiencies and off-target editing remain challenges for the field. Full article

Lettuce (Lactuca sativa L.) is an important leafy vegetable crop, yet the efficiency and reliability of genome editing platforms in lettuce remain limited, particularly for precision base editing applications. In this study, we established an optimized PEG-mediated protoplast transformation system for lettuce through systematic evaluation of key parameters, including protoplast density, incubation time, plasmid size, and transformation method. Under optimized conditions, a maximum transient transformation efficiency of up to 81% was achieved. Using this optimized protoplast platform, we comparatively evaluated the performance of three single-base editing systems—adenosine base editor (ABE), glycosylase-based guanine base editor (gGBE), and rice alkylpurine DNA glycosylase-mediated A-to-K base editor (rAKBE)—targeting the LsALS gene, encoding acetolactate synthetase as a herbicide target with great value in weed control. Among the tested editors, ABE exhibited the highest A-to-G editing efficiency, reaching 9.3%. In contrast, gGBE and rAKBE showed lower editing efficiencies. Together, this study established a robust and reproducible protoplast-based platform for transient genome editing in lettuce and provides a practical framework for the rapid evaluation of base editing tools and target sites, firstly for gGBE and rAKBE evaluation in lettuce. The optimized system facilitates functional genomics studies and supports the development of precision breeding strategies in lettuce. Full article

Cultivated lettuce (Lactuca sativa L.) is a major leafy crop and an emerging model for functional genomics within the Asteraceae family, supported by high-quality reference genomes and efficient transformation systems. Although CRISPR/Cas technology offers powerful opportunities for crop improvement, editing efficiency depends on optimized construct architecture and reliable guide RNA (gRNA) validation. However, a rapid platform for evaluating CRISPR reagents in lettuce is still lacking. Here, we developed an efficient hairyroot-based system to accelerate CRISPR/Cas genome editing optimization in L. sativa. Four Agrobacterium rhizogenes strains were compared for hairy root induction in two cultivars, ‘Saladin’ and ‘Osiride’, identifying strain ATCC15834 as the most effective based on transformation frequency and root production. Using this platform, we evaluated multiple CRISPR construct configurations, including alternative promoters for nuclease and gRNA expression. A plant-derived promoter combined with At-pU6-26 variant significantly improved editing efficiency. As a proof of concept, we targeted LsHB2, the putative ortholog of Arabidopsis thaliana ATHB2, a key regulator of the shade avoidance response using SpCas9, SaCas9, and LbCas12a nucleases. The system enabled rapid genotyping and quantitative indel profiling. Overall, this workflow provides a robust framework for efficient guide selection and construct optimization in lettuce genome editing. Full article

Plant viral vectors are powerful tools for the transient expression of exogenous genes, enabling not only virus-induced gene silencing (VIGS) but also virus-induced genome editing (VIGE). However, technical systems capable of simultaneously achieving gene silencing and gene editing in cotton have been rarely reported to date. Therefore, the development of a virus vector system that can concurrently mediate both gene editing and gene silencing would provide a valuable platform for advancing functional genomics studies and molecular design breeding in cotton. To address this gap, we established a system in cotton that concurrently enables gene silencing and gene editing. This system utilizes cotton Cas9 overexpression (Cas9-OE) as a receptor and CLCrV and TRV as vectors for targeting the GhCLA1 gene, which yields an albino phenotype upon silencing and mutation. Initially, CLCrV and TRV were used independently as vectors for gene editing and gene silencing, respectively. However, our results demonstrated persistent GhCLA1 gene silencing via TRV, but no systemic gene editing via CLCrV, suggesting viral cross-protection may occur between CLCrV and TRV for simultaneous actions. Subsequently, we constructed tandem assemblies of GhCLA1 silencing fragments and sgRNA expression elements in both TRV and CLCrV vectors resulted in successful gene silencing and editing, albeit with low editing efficiency. Further optimization through shortening the gene silencing fragments led to a substantial 2.61 to 3.11-fold increase in editing efficiency, while still maintaining effective GhCLA1 silencing. This refined system provides a robust tool for gene editing in cotton. Full article

Climate change has adversely affected grain production and quality of canola, the second-largest oilseed crop, which contributes 13–16% of total vegetable oil. Multiple biotic and abiotic stresses significantly limit canola production due to rapid climate change, and conventional breeding alone is insufficient to meet global demand. Therefore, several advanced biotechnologies have been developed to cope with this change. Among these, genetic modification, gene editing, and RNA interference are particularly significant for rapid cultivar development in a cost-effective, efficient, and convenient way. Recent findings in gene editing applications have revealed “prospective sites”, highlighting regions amenable to precise editing without compromising canola plant growth or development. Pan-genome analyses have further guided gene editing target selection, enabling the validation of key stress-resilience genes across diverse canola cultivars, while the CRISPR-epigenetic regulatory connection enables targeted control of gene expression and trait modulation. A hypothetical application of genomic selection is also suggested, which could complement gene editing to accelerate the development of superior cultivars. Accordingly, this review focuses on the latest studies of genetic modification, gene editing, and RNA interference to strengthen canola resilience under rapid climate change and discusses the major concerns. Taken together, these genome-editing strategies offer precise approaches for improving biotic and abiotic stress tolerance, although careful consideration of both off-target effects and regulatory compliance remains essential for their practical implementation in canola improvement. Full article

The artificially cultivated edible mushroom Stropharia rugosoannulata is widely promoted and cultivated in China because of its ability to efficiently decompose agricultural and forestry waste. However, methods for CRISPR/Cas9 genome editing have not yet been established for S. rugosoannulata. In this study, we identified three SrU6 promoters in S. rugosoannulata and constructed the CRISPR/Cas9 expression vector GPiE-SrU6. Moreover, we found that mutant strains were obtained only when the expression of the single guide RNA (sgRNA) was driven by the SrU6-3 promoter. We subsequently employed a tandemly repeated SrU6-tRNA-sgRNA module to knock out two sites within the ura3 gene. The expression vector was introduced into the mycelium via Agrobacterium-mediated transformation (ATMT). Following dual selection with 60 μg/mL hygromycin (Hyg) and 0.2 mg/mL 5-fluoroorotic acid (5-FOA), stable transformants were obtained and subcultured. The mutation efficiency at the targeted ura3 locus was subsequently assessed. The CRISPR/Cas9 system successfully disrupted the target marker gene (ura3), achieving an editing efficiency of 14.9%. In summary, this study reports the first successful establishment of a CRISPR/Cas9 genome editing system in S. rugosoannulata. This study not only meets a future need for genetic manipulation tools for S. rugosoannulata but also provides a robust platform for engineering superior strains for eco-circular agriculture. Full article

Stevia rebaudiana Bertoni is a strategically important perennial crop because it is the main botanical source of steviol glycosides, a group of high-intensity, non-caloric sweeteners increasingly demanded by the global food and beverage industry. Despite the rapid expansion of stevia cultivation, commercial production remains strongly dependent on a narrow genetic base, particularly on clonally propagated cultivars such as ‘Morita II’, which has long served as the industrial benchmark because of its favourable rebaudioside A profile and processing consistency. This dependence has raised concerns about limited adaptive capacity, genetic erosion and restricted long-term breeding progress. In this review, we provide an integrated and critical synthesis of current knowledge on the genetic diversity of S. rebaudiana, the biosynthetic and regulatory architecture of steviol glycosides, and the conventional and emerging strategies available for crop improvement. Unlike previous reviews, this article explicitly connects domestication-driven genetic bottlenecks, wild germplasm mobilisation, metabolic pathway regulation, advanced analytical phenotyping and precision breeding into a single systems-oriented framework. We examine the roles of wild germplasm, somaclonal variation, polyploidy, molecular markers, omics-assisted approaches and transgene-free genome editing as complementary tools to broaden the stevia breeding base while preserving industrial quality standards. We finally propose an integrative roadmap for the sustainable genetic improvement of stevia, positioning ‘Morita II’ not as an endpoint, but as a benchmark within a broader diversification strategy. Full article

The SHAGGY-like kinase (SK) gene family regulates diverse developmental and abiotic stress response processes in plants. Although genome-wide analyses of SKs have been conducted in model plants such as Arabidopsis thaliana and rice, their characterization in the economically important crop Brassica rapa remains limited. In this study, we conducted a systematic genome-wide analysis of SK genes in three Brassica species. A total of 18, 16, and 18 SK members were identified in B. rapa, B. nigra, and B. oleracea, respectively, and phylogenetic analysis classified them into four distinct clades. Expression profiling revealed that BrSKβ-1 and BrSKβ-2 were specifically expressed in fertile floral buds, suggesting their critical roles in pollen development. Furthermore, co-expression analysis indicated that both genes were co-expressed with key regulators involved in pollen development, pollen sperm cell differentiation and pollen tube growth. Loss of BrSKβ-2 via CRISPR/Cas9 resulted in 25–65% pollen abnormality and reduced the germination rate of normal-appearing pollen to only 10%, confirming its essential role in male fertility. Together, these findings provide a comprehensive characterization of the SK gene family in Brassica and position BrSKβ-2 as a promising candidate for gene editing-based male sterility systems in B. rapa and related crops. Full article

Background/Objectives: Castanopsis tibetana Hance (C. tibetana) is a valuable timber species in southern China. Its chloroplast and nuclear genomes have been characterized, but its mitochondrial genome (mitogenome) remains unknown. This study assembles and characterizes the first complete mitogenome of C. tibetana, elucidating its structural and evolutionary features. Methods: A hybrid approach combining Oxford Nanopore long reads and Illumina short reads was used. The mitogenome was assembled via iterative seed-based mapping and annotated via GeSeq and tRNAscan-SE. Repeats were identified via MISA, TRF, and REPuter. The RNA editing sites were predicted with the PREP suite. Phylogenetic analysis was performed on 14 conserved protein-coding genes from 13 species via maximum likelihood and Bayesian inference. Results: The mitogenome is a 554,078 bp circular molecule (GC 45.27%) encoding 51 genes (32 PCGs, 16 tRNAs, 3 rRNAs). It contains 202 simple sequence repeats (37.1% tetrameric). We predicted 53 C-to-U RNA editing sites, most frequently in nad7 and nad5. Codon usage showed bias, with 28 codons having RSCU > 1. Twenty fragments (6001 bp, 1.08% of the mitogenome) were transferred from the chloroplast. Phylogenomic analysis placed C. tibetana within Fagaceae, close to other Castanopsis species. Conclusions: This study provides the first comprehensive characterization of the C. tibetana mitogenome, revealing its structural architecture, repetitive landscape, RNA editing profile, and phylogenetic placement. These findings offer valuable genomic resources for understanding mitogenome evolution in Fagaceae and support future research on the conservation genetics and molecular breeding of this important tree species. Full article

Pedicularis henryi is a hemiparasitic species within Orobanchaceae (Lamiales). In this study, the mitochondrial genome of P. henryi was assembled and characterized. The mitogenome is 251,317 bp in length with a GC content of 44.32%, containing 36 protein-coding genes, 24 tRNAs, and three rRNAs. Codon usage analysis revealed a marked preference for A/U-terminated codons. A total of 196 repetitive elements were identified, with interspersed repeats as the most abundant type. We detected 293 C-to-U RNA editing sites across 31 protein-coding genes, predominantly causing non-synonymous substitutions. Eighteen chloroplast-derived fragments totaling 35,894 bp were found, accounting for 15.0% of the mitogenome. Nucleotide diversity analysis among three Pedicularis species showed an average π of 0.0018, with core respiratory genes highly conserved. Synteny analysis revealed extensive structural rearrangement in P. henryi compared to P. chinensis. This study provides mitochondrial genomic resources for Orobanchaceae and insights into mitogenome evolution in hemiparasitic plants. Full article

The eukaryotic translation initiation factor 4E (eIF4E) family plays a dual role in plants, regulating cap-dependent protein synthesis and mediating susceptibility to viruses in the family Potyviridae. In cowpea (Vigna unguiculata (L.) Walp.), an economically important legume cultivated worldwide, the structural determinants of these isoforms remain largely unexplored. This study characterizes the genomic organization, evolutionary history, and conformational dynamics of eIF4E, eIF(iso)4E, and nCBP in cowpea using a multi-omics approach. Genome mining identified three paralogous genes located on chromosomes 4, 6, and 7, showing high synteny with Phaseolus vulgaris. Phylogenetic analysis confirmed nCBP as the ancestral Class I lineage, distinct from the Class II eIF4E and eIF(iso)4E clades. Theoretical models for the isoforms were generated and subsequently validated by molecular dynamics simulations, revealing that while all isoforms preserve the canonical tertiary architecture and an electropositive cap-binding pocket, eIF(iso)4E exhibits superior structural compactness and hydrogen-bond stability. These biophysical features highlight their role as a stable anchor for viral VPg proteins. By elucidating the atomic-level landscape of these factors, we provide a robust structural framework to guide allele mining and genome-editing strategies aiming to engineer virus-resistant cowpea cultivars without compromising agronomic performance. Full article

Cutaneous T-cell lymphoma (CTCL) comprises a heterogeneous group of extranodal non-Hodgkin lymphomas. With the publication of the fifth edition of the World Health Organization Classification of Hematolymphoid Tumors, the diagnostic framework for CTCL has shifted from primarily morphologic phenotypes toward an emphasis on molecular drivers. Current research suggests that malignant clones may arise from somatic mutations at the hematopoietic stem cell stage and may follow a continuous hematogenous dissemination model with bidirectional trafficking between the skin and systemic circulation. At the molecular level, genomic instability, often associated with somatic copy-number variations, may promote activation of the janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway through gene-dosage effects. In parallel, chromatin remodeling linked to EZH2 overexpression and reduced special SATB1 expression may support a Th2-polarized program. This phenotype may contribute to epidermal barrier impairment via cytokines such as Interleukins-4 (IL-4) and IL-13, potentially creating conditions permissive for Staphylococcus aureus colonization. Microbial superantigens and exotoxins may further contribute to tumor progression and therapeutic resistance by reinforcing JAK/STAT signaling, particularly STAT3, and reducing CD8+ T-cell–mediated immune surveillance. In the dermis, reprogramming of cancer-associated fibroblasts and polarization of macrophages toward an M2 phenotype may collectively contribute to an immunosuppressive niche. Emerging biomarkers, including CD74, and acquired resistance mechanisms after anti-C-C chemokine receptor 4 therapy further extend the translational relevance of recent pathologic findings. Overall, CTCL evolution appears to be a systemic process shaped by interactions between tumor-intrinsic genetic alterations and the skin microenvironment. Full article

CRISPR–Cas9 has progressed from an experimental tool to a therapeutic modality, marked by the first regulatory approvals of an ex vivo-edited autologous CD34+ hematopoietic stem cell product that induces fetal hemoglobin (CASGEVY/exa-cel). In this narrative review, we synthesize modality-specific molecular diagnostic strategies used across early CRISPR clinical translation. In parallel, early clinical experience has begun to demonstrate the feasibility of in vivo editing, including subretinal delivery for CEP290-associated inherited retinal degeneration (EDIT-101 programme) and hepatocyte-targeted lipid nanoparticles (LNPs) for liver-derived targets such as transthyretin and plasma prekallikrein (KLKB1). As translation expands across hematologic, metabolic, ocular and oncology indications, development is increasingly constrained by the predictability and safety of editing outcomes, delivery-determined biodistribution and exposure time, and immune recognition of bacterial Cas9 orthologs and delivery components. We summarize diagnostic readouts for confirming patient genotype, quantifying on-target editing and expression changes, assessing off-target and structural outcomes using orthogonal assays, and monitoring clonal dynamics and immune responses during long-term follow-up. We also discuss how these readouts interface with CMC controls and regulatory expectations for advanced therapy medicinal products (ATMPs), highlighting the need for fit-for-purpose, standardized testing frameworks in early trials. Full article

A simple method for the efficient high-throughput transformation of hybrid poplar (Populus tremula x alba clone 717 1B) is described. Factors considered in developing the method included the ease and efficiency of preparing large numbers of explants for transformation, and selection of culture media that enhanced cell and tissue growth while promoting shoot regeneration competence. We found that petiole explants from in vitro-cultured plantlets can be easily collected and prepared for transformation and regenerate shoots comparable to stem or leaf explants. Culturing petiole explants on Driver Kuniyuki Walnut (DKW) medium resulted in significantly greater tissue growth compared to Murashige Skoog (MS) medium. Moreover, the inclusion of low concentrations of thidiazuron (TDZ) in callus-inducing media (CIM) significantly enhanced shoot regeneration competence of cultured petiole explants. As a consequence, the combination of petiole explants cultured on DKW medium along with 2.2 ug/L TDZ during the callus induction phase resulted in rapid and efficient transformation of this hybrid poplar genotype. When applied to genomic approaches such as activation tagging or CRISPR-Cas9 and Cas12a gene editing, we obtained transformation efficiencies ranging between 70% and 90%. The described protocol provides a simple and efficient method that is easily scalable for high-throughput approaches, which could facilitate genome-wide methods for the rapid and efficient production of transformed hybrid poplars. Full article