Search Results (641)

Plant architecture is a crucial agronomic trait significantly impacting soybean (Glycine max) yield. Traditional breeding has made some progress in optimizing soybean architecture, but it is limited in precision and efficiency. The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein (CRISPR/Cas) system, a revolutionary gene-editing technology, provides unprecedented opportunities for plant genetic improvement. This review outlines CRISPR’s development and applications in crop improvement, focusing specifically on progress regulating soybean architecture traits affecting yield, such as node number, internode length, branching, and leaf morphology. It also discusses the technical challenges for CRISPR technology in enhancing soybean architecture, including that the regulatory network of soybean plant architecture is complex and the development of multi-omics platforms helps gene mining. The application of CRISPR enables precise the regulation of gene expression through promoter editing. Meanwhile, it is also faced with technical challenges such as the editing of homologous genes caused by genome polyploidy, the efficiency of editing tools and off-target effects, and low transformation efficiency. New delivery systems such as virus-induced genome editing bring hope for solving some of these problems. The review emphasizes the great potential of CRISPR technology in breeding next-generation soybean varieties with optimized architecture to boost yield potential. Full article

Hippophae rhamnoides ssp. sinensis Rousi a species of significant ecological and economic value that is native to the Qinghai–Tibet Plateau and arid/semi-arid regions. Investigating the mitochondrial genome can elucidate stress adaptation mechanisms, population genetic structure, and hybrid evolutionary history, offering molecular insights for ecological restoration and species conservation. However, the genetic information and evolutionary mechanisms of its mitochondrial genome remain poorly understood. This study aimed to assemble the complete mitochondrial genome of H. rhamnoides L. ssp. sinensis using Illumina sequencing, uncovering its structural features, evolutionary pressures, and environmental adaptability and addressing the research gap regarding mitochondrial genomes within the Hippophae genus. The study assembled a 454,444 bp circular mitochondrial genome of H. rhamnoides ssp. sinensis, with a GC content of 44.86%. A total of 73 genes and 3 pseudogenes were annotated, with the notable absence of the rps2 gene, which is present in related species. The genome exhibits significant codon usage bias, particularly with high-frequency use of the alanine codon GCU and the isoleucine codon AUU. Additionally, 449 repetitive sequences, potentially driving genome recombination, were identified. Our evolutionary pressure analysis revealed that most genes are under purifying selection, while genes such as atp4 and nad4 exhibit positive selection. A nucleotide diversity analysis revealed that the sdh4 gene exhibits the highest variation, whereas rrn5 is the most conserved. Meanwhile, phylogenetic analysis showed that H. rhamnoides ssp. sinensis from China is most closely related to Hippophae tibetana, with extensive homologous sequences (49.72% of the chloroplast genome) being identified between the chloroplast and mitochondrial genomes, indicating active inter-organellar gene transfer. Furthermore, 539 RNA editing sites, primarily involving hydrophilic-to-hydrophobic amino acid conversions, were predicted, potentially regulating mitochondrial protein function. Our findings establish a foundation for genetic improvement and research on adaptive evolutionary mechanisms in the Hippophae genus, offering a novel case study for plant mitochondrial genome evolution theory. Full article

Plants must contend with oxidative stress, a paradoxical phenomenon in which reactive oxygen species (ROS) can cause cellular damage while also serving as key signaling molecules. Environmental stressors, such as drought, salinity, and temperature extremes, promote ROS accumulation, affecting plant growth and productivity. To maintain redox homeostasis, plants rely on antioxidant systems comprising enzymatic defenses, such as superoxide dismutase, catalase, and ascorbate peroxidase, and non-enzymatic molecules, including ascorbate, glutathione, flavonoids, and emerging compounds such as proline and nano-silicon. This review provides an integrated overview of antioxidant responses and their modulation through recent biotechnological advances, emphasizing the role of emerging technologies in advancing our understanding of redox regulation and translating molecular insights into stress-resilient phenotypes. Omics approaches have enabled the identification of redox-related genes, while genome editing tools, particularly those based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, offer opportunities for precise functional manipulation. Artificial intelligence and systems biology are accelerating the discovery of regulatory modules and enabling predictive modeling of antioxidant networks. We also highlight the contribution of synthetic biology to the development of stress-responsive gene circuits and address current regulatory and ethical considerations. Overall, this review aims to provide a comprehensive perspective on molecular, biochemical, and technological strategies to enhance oxidative stress tolerance in plants, thereby contributing to sustainable agriculture and food security in a changing climate. Full article

Background and Objectives: Over the past few years, there has been a significant shift in focus from developing better diagnostic tools to detecting Alzheimer’s disease (AD) earlier and initiating treatment interventions. This review will explore four main objectives: (a) the role of biomarkers in enhancing the diagnostic accuracy of AD, highlighting the major strides that have been made in recent years; (b) the role of neuropsychological testing in identifying biomarkers of AD, including the relationship between cognitive performance and neuroimaging biomarkers; (c) the amyloid hypothesis and possible molecular mechanisms of AD; and (d) the innovative AD therapeutics and the challenges and limitations of AD research. Materials and Methods: We have searched PubMed and Scopus databases for peer-reviewed research articles published in English (preclinical and clinical studies as well as relevant reviews and meta-analyses) investigating the molecular mechanisms, biomarkers, and treatments of AD. Results: Genome-wide association studies (GWASs) discovered 37 loci associated with AD risk. Core 1 biomarkers (α-amyloid Aβ42, phosphorylated tau, and amyloid PET) detect early AD phases, identifying both symptomatic and asymptomatic individuals, while core 2 biomarkers inform the short-term progression risk in individuals without symptoms. The recurrent failures of Aβ-targeted clinical studies undermine the amyloid cascade hypothesis and the objectives of AD medication development. The molecular mechanisms of AD include the accumulation of amyloid plaques and tau protein, vascular dysfunction, neuroinflammation, oxidative stress, and lipid metabolism dysregulation. Significant advancements in drug delivery technologies, such as focused Low-Ultrasound Stem, T cells, exosomes, nanoparticles, transferin, nicotinic and acetylcholine receptors, and glutathione transporters, are aimed at overcoming the BBB to enhance treatment efficacy for AD. Aducanumab and Lecanemab are IgG1 monoclonal antibodies that retard the progression of AD. BACE inhibitors have been explored as a therapeutic strategy for AD. Gene therapies targeting APOE using the CRISPR/Cas9 genome-editing system are another therapeutic avenue. Conclusions: Classic neurodegenerative biomarkers have emerged as powerful tools for enhancing the diagnostic accuracy of AD. Despite the supporting evidence, the amyloid hypothesis has several unresolved issues. Novel monoclonal antibodies may halt the AD course. Advances in delivery systems across the BBB are promising for the efficacy of AD treatments. Full article

Background: Centella asiatica, a medicinally important species that is rich in bioactive compounds, lacks a characterized mitochondrial genome, despite nuclear and chloroplast assemblies. We sequenced and annotated its mitochondrial genome to elucidate its genetic foundations and evolutionary mechanisms. Methods: Assembly using Illumina short-reads and Nanopore long-reads was used to characterize the mitochondrial genome. Analyses included structural characterization, codon usage bias, repetitive sequences, horizontal gene transfer (HGT), collinearity, and phylogeny. The resulting tissue-specific (root, stem, and leaf) long non-coding RNA (lncRNA) profiles identified RNA editing sites. Results: The complete mitochondrial genome (249,777 bp, 45.5% GC) comprises three circular contigs encoding 51 genes (33 protein-coding, 15 tRNA, and 3 rRNA). Comparative genomics revealed synteny with the Apiaceae family of plants and evidence of HGT. Phylogenetic analysis resolved taxonomic relationships within Apiales. We predicted that 547 RNA editing sites would be identified in its protein-coding genes. Tissue profiling identified 725 (root), 711 (stem), and 668 (leaf) editing sites, with >71% concordance to predictions. RNA editing-generated cryptic promoters/terminators occur in mitochondrial core function genes (e.g., ATP synthase, cytochrome c reductase/oxidase, ribosome large subunit, and cytochrome c biogenesis), exhibiting a lower frequency in the leaves compared to the roots and stems. Conclusions: We provide the first complete mitochondrial genome assembly for C. asiatica, delineating its complex structure, tissue-modulated RNA editing, and evolutionary trajectory. This high-quality genomic resource establishes a foundation for molecular evolutionary studies and enhances the genomic toolkit for this pharmacologically significant species. Full article

Retinitis pigmentosa is a group of inherited retinal dystrophies characterized by progressive photoreceptor cell loss leading to irreversible vision loss. Affecting approximately 1 in 4000 individuals worldwide, retinitis pigmentosa exhibits significant genetic heterogeneity, with mutations in genes such as RHO, PRPF31, RPE65, USH2A, and NR2E3, which contribute to its diverse clinical presentation. This review outlines the genetic basis of retinitis pigmentosa and explores cutting-edge gene-based therapeutic strategies. Luxturna (voretigene neparvovec-rzyl), the first FDA-approved gene therapy targeting RPE65 mutations, represents a milestone in precision ophthalmology, while OCU400 is a gene-independent therapy that uses a modified NR2E3 construct to modulate retinal homeostasis across different RP genotypes. Additionally, CRISPR–Cas genome-editing technologies offer future potential for the personalized correction of specific mutations, though concerns about off-target effects and delivery challenges remain. The article also highlights MCO-010, a novel optogenetic therapy that bypasses defective phototransduction pathways, showing promise for patients regardless of their genetic profile. Moreover, QR-1123, a mutation-specific antisense oligonucleotide targeting the P23H variant in the RHO gene, is under clinical investigation for autosomal dominant RP and has shown encouraging preclinical results in reducing toxic protein accumulation and preserving photoreceptors. SPVN06, another promising candidate, is a mutation-agnostic gene therapy delivering RdCVF and RdCVFL via AAV to support cone viability and delay degeneration, currently being evaluated in a multicenter Phase I/II trial for patients with various rod–cone dystrophies. Collectively, these advances illustrate the transition from symptom management toward targeted, mutation-specific therapies, marking a major advancement in the treatment of RP and inherited retinal diseases. Full article

Prime editing (PE) has emerged as a transformative genome editing technology, enabling precise base substitutions, insertions, and deletions without inducing double-strand DNA breaks (DSBs). However, its application in zebrafish remains limited by low efficiency. Here, we leveraged PE7, a state-of-the-art PE system, combined with La-accessible prime editing guide RNAs (pegRNAs), to enhance editing efficiency in zebrafish. By co-incubating PE7 protein with La-accessible pegRNAs to form ribonucleoprotein (RNP) complexes and microinjecting these complexes into zebrafish embryos, we achieved up to 15.99% editing efficiency at target loci—an improvement of 6.81- to 11.46-fold over PE2. Additionally, we observed 16.60% 6 bp insertions and 13.18% 10 bp deletions at the adgrf3b locus, representing a 3.13-fold increase over PE2. Finally, we used PE to introduce desired edits at the tyr locus, successfully generating zebrafish with the tyr P302L mutation that exhibited melanin reduction. These findings demonstrate that PE7 significantly enhances prime editing efficiency in fish, providing novel tools for functional gene studies and genetic breeding in aquatic species. Full article

Rice seed storage proteins (SSPs) play a critical role in determining the nutritional quality, cooking properties, and digestibility of rice. To enhance seed quality, CRISPR/Cas9 genome editing was applied to modify SSP composition by targeting genes encoding 13 kDa prolamins and type A glutelins. Three CRISPR/Cas9 constructs were designed: one specific to the 13 kDa prolamin subfamily and two targeting conserved GluA glutelin regions. Edited T₀ and T₁ lines were generated and analyzed using InDel analysis, SDS-PAGE, Bradford assay, and RP-HPLC. Insertions were more frequent than deletions, accounting for 56% and 74% of mutations in prolamin and glutelin genes, respectively. Editing efficiency varied between sgRNAs. All lines with altered protein profiles contained InDels in target genes. SDS-PAGE confirmed the absence or reduction in bands corresponding to 13 kDa prolamins or GluA subunits, showing consistent profiles among lines carrying the same construct. Quantification revealed significant shifts in SSP composition, including increased albumin and globulin content. Prolamin-deficient lines showed reduced prolamins, while GluA-deficient lines exhibited increased prolamins. Total protein content was significantly elevated in all edited lines, suggesting enrichment in lysine-rich fractions. These findings demonstrate that CRISPR/Cas9-mediated editing of SSP genes can effectively reconfigure the rice protein profile and enhance its nutritional value. Full article

Genetic transformation is an essential tool for investigating gene function and editing genomes. Kiwifruit, recognized as a significant global fresh fruit crop, holds considerable economic and nutritional importance. However, current genetic transformation techniques for kiwifruit are impeded by low efficiency, lengthy culture durations (a minimum of six months), and substantial labor requirements. In this research, we established an efficient system for shoot regeneration and the stable genetic transformation of the ‘Hayward’ cultivar, utilizing leaf explants in conjunction with two strains of Agrobacterium that harbor the expression vector pBI121-35S::GFP, which contains the green fluorescent protein (GFP) gene as a visible marker within the T-DNA region. Our results show that 93.3% of leaf explants responded positively to the regeneration medium, producing multiple independent adventitious shoots around the explants within a six-week period. Furthermore, over 71% of kanamycin-resistant plantlets exhibited robust GFP expression, and the entire transformation process was completed within four months of culture. Southern blot analysis confirmed the stable integration of GFP into the genome, while RT-PCR and fluorescence microscopy validated the sustained expression of GFP in mature plants. This efficient protocol for regeneration and transformation provides a solid foundation for micropropagation and the enhancement of desirable traits in kiwifruit through overexpression and gene silencing techniques. Full article

Legumes are vital sources of protein, dietary fibre and nutrients, making them crucial for global food security and sustainable agriculture. However, their widespread acceptance and consumption are often limited by undesirable sensory characteristics, such as “a beany flavour”, bitterness or variable textures. Addressing these challenges requires a comprehensive understanding of the complex molecular mechanisms governing appearance, aroma, taste, flavour, texture and palatability in legumes, aiming to enhance their sensory appeal. This review highlights the transformative power of multi-omics approaches in dissecting these intricate biological pathways and facilitating the targeted enhancement of legume sensory qualities. By integrating data from genomics, transcriptomics, proteomics and metabolomics, the genetic and biochemical networks that directly dictate sensory perception can be comprehensively unveiled. The insights gained from these integrated multi-omics studies are proving instrumental in developing strategies for sensory enhancement. They enable the identification of key biomarkers for desirable traits, facilitating more efficient marker-assisted selection (MAS) and genomic selection (GS) in breeding programs. Furthermore, a molecular understanding of sensory pathways opens avenues for precise gene editing (e.g., using CRISPR-Cas9) to modify specific genes, reduce off-flavour compounds or optimise texture. Beyond genetic improvements, multi-omics data also inform the optimisation of post-harvest handling and processing methods (e.g., germination and fermentation) to enhance desirable sensory profiles and mitigate undesirable ones. This holistic approach, spanning from the genetic blueprint to the final sensory experience, will accelerate the development of new legume cultivars and products with enhanced palatability, thereby fostering increased consumption and ultimately contributing to healthier diets and more resilient food systems worldwide. Full article

The recent discovery of TIGR-Tas (Tandem Interspaced Guide RNA-Targeting Systems) marks a major advance in the field of genome editing, introducing a new class of compact, programmable DNA-targeting systems that function independently of traditional CRISPR-Cas pathways. TIGR-Tas effectors use a novel dual-spacer guide RNA (tigRNA) to recognize both strands of target DNA without requiring a protospacer adjacent motif (PAM). These Tas proteins introduce double-stranded DNA cuts with characteristic 8-nucleotide 3′ overhangs and are significantly smaller than Cas9, offering delivery advantages for in vivo editing. Structural analyses reveal homology to box C/D snoRNP proteins, suggesting a previously unrecognized evolutionary lineage of RNA-guided nucleases. This review positions TIGR-Tas at the forefront of a new wave of RNA-programmable genome-editing technologies. In parallel, I provide comparative insight into the diverse and increasingly modular CRISPR-Cas systems, including Cas9, Cas12, Cas13, and emerging effectors like Cas3, Cas10, CasΦ, and Cas14. While the CRISPR-Cas universe has revolutionized molecular biology, TIGR-Tas systems open a complementary and potentially more versatile path for programmable genome manipulation. I discuss mechanistic distinctions, evolutionary implications, and potential applications in human cells, synthetic biology, and therapeutic genome engineering. Full article

The protein p53, often referred to as the “guardian of the genome,” is essential for preserving cellular balance and preventing cancerous transformations. As one of the most commonly altered genes in human cancers, its impaired function is associated with tumor initiation, development, and resistance to treatment. Exploring the diverse roles of p53, which include regulating the cell cycle, repairing DNA, inducing apoptosis, reprogramming metabolism, and modulating immunity, provides valuable insights into cancer mechanisms and potential treatments. This review integrates recent findings on p53′s dual nature, functioning as both a tumor suppressor and an oncogenic promoter, depending on the context. Wild-type p53 suppresses tumors by inducing cell cycle arrest or apoptosis in response to genotoxic stress, while mutated variants often lose these functions or gain novel pro-oncogenic activities. Emerging evidence highlights p53′s involvement in non-canonical pathways, such as regulating tumor microenvironment interactions, metabolic flexibility, and immune evasion mechanisms. For instance, p53 modulates immune checkpoint expression and influences the efficacy of immunotherapies, including PD-1/PD-L1 blockade. Furthermore, advancements in precision diagnostics, such as liquid biopsy-based detection of p53 mutations and AI-driven bioinformatics tools, enable early cancer identification and stratification of patients likely to benefit from targeted therapies. Therapeutic strategies targeting p53 pathways are rapidly evolving. Small molecules restoring wild-type p53 activity or disrupting mutant p53 interactions, such as APR-246 and MDM2 inhibitors, show promise in clinical trials. Combination approaches integrating gene editing with synthetic lethal strategies aim to exploit p53-dependent vulnerabilities. Additionally, leveraging p53′s immunomodulatory effects through vaccine development or adjuvants may enhance immunotherapy responses. In conclusion, deciphering p53′s complex biology underscores its unparalleled potential as a biomarker and therapeutic target. Integrating multi-omics analyses, functional genomic screens, and real-world clinical data will accelerate the translation of p53-focused research into precision oncology breakthroughs, ultimately improving patient outcomes. Full article

Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription–translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology. Full article

Lysosomal storage disorders (LSDs) are inherited metabolic conditions characterized by lysosomal enzyme deficiencies leading to substrate accumulation. As genetic diseases, LSDs can be treated with gene therapies (GT), including the CRISPR/Cas systems. The CRISPR/Cas systems enable precise and programmable genome editing, leading to targeted modifications at specific genomic loci. While the classical CRISPR/Cas9 system has been extensively used to generate LSD disease models and correct disease-associated genetic alterations through homologous recombination (HR), recently described Cas proteins as well as CRISPR/Cas9-derived strategies such as base editing, prime editing, and homology-independent targeted integration (HITI) offer a novel way to develop innovative treatments for LSDs. The direct administration of the CRISPR/Cas9 system remains the primary strategy evaluated in several LSDs; nevertheless, the ex vivo CRISPR/Cas9-based approach has been recently explored, primarily in central nervous system-affecting LSDs. Ex vivo approaches involve genetically modifying, in theory, any patient cells in the laboratory and reintroducing them into the patient to provide a therapeutic effect. This manuscript reviews the molecular aspects of the CRISPR/Cas technology and its implementation in ex vivo strategies for LSDs while discussing novel approaches beyond the classical CRISPR/Cas9 system. Full article

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering. Full article