Search Results (1,571)

Myotonic Dystrophy Type 1 (DM1) is a complex multisystemic genetic disorder caused by CTG repeat expansions in the DMPK gene, leading to RNA toxicity and widespread splicing defects. These splicing abnormalities affect multiple systems, including the respiratory, skeletal, cardiac, nervous, and endocrine systems, resulting in aggressive symptoms that significantly impact quality of life and survival. Cardiac complications are the second leading cause of deaths in DM1, after respiratory insufficiency. Current research is largely focused on understanding cardiac pathology in DM1. This review highlights recent advancements in the clinical and pathological characterization of DM1 cardiac involvement, preclinical models used to study cardiac dysfunction, and emerging therapeutic strategies that target the molecular basis of DM1. Promising approaches include RNA-targeting strategies such as antisense oligonucleotides (ASOs), gene-editing tools like CRISPR-Cas9, and small molecules that modulate RNA splicing. ASOs aim to reduce toxic RNA accumulation, CRISPR-based approaches aim to excise or correct the expanded CTG repeats, and repurposed small-molecule drugs, such as vorinostat, tideglusib, and metformin, could serve as potential therapeutic agents for DM1 patients with cardiac complications. Despite this progress, several challenges remain: the heterogeneity of cardiac manifestations, unpredictable and often silent progression of arrhythmias, limited therapeutic options beyond implantable cardioverter-defibrillator (ICD)/pacemaker implantations, and complex interplay with the multisystemic nature of DM1. More research and well-designed clinical trials are urgently needed to translate these promising strategies into effective treatments for DM1-associated cardiac disease. Here, we discuss the current knowledge in DM1 cardiac pathology and preclinical models as well as the benefits and pitfalls of the available therapeutic approaches. Full article

African swine fever (ASF), induced by the African swine fever virus (ASFV), is an acute hemorrhagic disease characterized by high fever, systemic hemorrhages, and elevated mortality. Current diagnostic techniques including PCR and ELISA present limitations in field applications due to requirements for specialized equipment and prolonged processing duration. Therefore, rapid and accurate detection of ASFV has become a key link in ASF prevention and control. This study established a rapid and precise visual diagnostic approach by integrating the CRISPR/AapCas12b system with lateral flow strip (LFS) technology, specifically targeting the B646L gene encoding the major capsid protein p72. The CRISPR/AapCas12b-LFS platform achieved a sensitivity threshold of 6 copies/µL for B646L gene detection, completing analysis within an hour. Validation study confirmed exceptional specificity against common porcine pathogens including PRRSV, CSFV, PRV, PPV4, and PCV3. The developed assay demonstrated complete concordance with real-time PCR results when analyzing 34 clinical specimens including three heart samples, three liver samples, three spleen samples, three lung samples, three kidney samples, three lymph node samples, five serum samples, five blood samples, and five oral swab samples for ASFV detection. Overall, this method is sensitive, specific, and practicable onsite for ASFV detection, showing a great application potential for monitoring ASFV in the field. Full article

Background/Objectives: Allele-specific genome editing using the CRISPR–Cas9 system is crucial for achieving precise therapeutic interventions in dominant inherited diseases that are otherwise difficult to treat with conventional approaches. However, Cas9–guide RNA (gRNA) complexes often tolerate single-base mismatches in target sequences, making it challenging to discriminate between wild-type and mutant alleles differing by only one nucleotide. Although previous studies have attempted to improve specificity by introducing mismatches into gRNAs, none has systematically investigated the impact of different mismatch types and positions on editing outcomes. In this study, we developed an effective strategy to enhance specificity and minimize off-target effects by deliberately introducing mismatches into gRNAs and comprehensively evaluating their editing performance. Results: We established an efficient strategy for the selective editing of mutant alleles that reduces Cas9 sequence tolerance and enhances specificity through the intentional introduction of mismatches into gRNAs. The efficacy of this approach was demonstrated by successful allele-specific editing of cancer-associated heterozygous point mutations in EGFR L858R and KRAS G12V, while minimizing editing of the corresponding wild-type alleles. Conclusion: Compared with perfectly matched gRNAs, the strategic incorporation of mismatches into gRNAs enhanced editing specificity for single-base mutant alleles. Our findings substantially improve the precision and safety of CRISPR-based genome editing for cancer therapy, particularly in cases involving mutant alleles. Full article

Tuberculosis (TB) remains a significant worldwide health challenge due to the limitations of conventional treatments and the rising incidence of drug-resistant Mycobacterium tuberculosis strains. This review consolidates the advancements in nanotechnology-based therapeutics, inhalable formulations, CRISPR–Cas tools, host-directed therapies (HDTs), and nanoparticle-based vaccine development aimed at enhancing TB management. Novel nanocarriers such as liposomes, solid-lipid nanoparticles (SLNs), dendrimers, and polymeric nanoparticles (NPs) offer enhanced bioavailability of drugs, sustained release, as well as targeted delivery to infected macrophages, thereby reducing systemic toxicity and dosing frequency. Inhalable nanomedicines provide localized delivery to the pulmonary site, enhancing the concentration of the drug at the primary site of infection. CRISPR–Cas technology is emerging as a transformative approach to disabling drug-resistant genes and enhancing diagnostic precision. HDTs, including agents like vitamin D and metformin, show potential in modulating host immune responses and enhancing pathogen clearance. Nanoparticle-based vaccines, including mRNA and antigen-conjugated platforms, aim to overcome the limitations of the BCG vaccine by enhancing antigen presentation and eliciting stronger, longer-lasting immunity. Collectively, these modalities mark a shift toward more personalized, effective, and less toxic TB therapies. However, challenges such as regulatory approval, safety, scalability, and accessibility remain. This review highlights the integrated potential of nanomedicine, gene editing, and immunomodulation to transform TB care and combat drug resistance, paving the way for more robust and durable treatment strategies. Full article

RNA methylation, particularly N6-methyladenosine (m⁶A) and 5-methylcytosine (m⁵C), functions as a pivotal post-transcriptional regulatory mechanism and plays a central role in plant growth, development, and stress responses. This review provides a systematic summary of recent advances in RNA methylation research in cucurbit crops. To date, high-throughput technologies such as MeRIP-seq and nanopore direct RNA sequencing have enabled the preliminary construction of RNA methylation landscapes in cucurbit species, revealing their potential regulatory roles in key agronomic traits, including fruit development, responses to biotic and abiotic stresses, and disease resistance. Nevertheless, this field remains in its early stages for cucurbit crops and faces several major challenges: First, mechanistic understanding is still limited, with insufficient knowledge regarding the composition and biological functions of the core protein families involved in methylation dynamics—namely, “writers,” “erasers,” and “readers.” Second, functional validation remains inadequate, as direct evidence linking specific RNA methylation events to downstream gene regulation and phenotypic outcomes is largely lacking. Third, resources are scarce; compared to model species such as Arabidopsis thaliana and rice, cucurbit crops possess limited species-specific genetic data and genetic engineering tools (e.g., CRISPR/Cas9-based gene editing systems), which significantly hampers comprehensive functional studies. To overcome these limitations, future research should prioritize the development and application of more sensitive detection methods, integrate multi-omics datasets—including transcriptomic and methylomic profiles—to reconstruct regulatory networks, and conduct rigorous functional assays to establish causal relationships between RNA methylation modifications and phenotypic variation. The ultimate objective is to fully elucidate the biological significance of RNA methylation in cucurbit plants and harness its potential for crop improvement through genetic and biotechnological approaches. Full article

In recent years, CRISPR-Cas9 technology has become a powerful and indispensable tool for targeted mutagenesis in plants, including applications such as gene knockout, prime-editing, multiplex gene editing, and regulation of gene transcription. As the number of potential genome editing approaches expands at a very fast pace, rapid, efficient, and cost-effective analytical strategies are needed to screen large numbers of mutants, including the detection of off-target events. In this study, we reported a detection method based on High-Resolution Melting (HRM) analysis to discriminate between wild-type (wt) and edited lines of different varieties of Vitis vinifera and grapevine rootstocks. Those edited lines were obtained through Agrobacterium tumefaciens mediated transformation of embryogenic calli using the CRISPR/SpCas9 system and targeting VviEPFL9-1 and VviEPFL9-2, two paralogous genes involved in stomata cell fate induction. The method clearly distinguished between the wt allele and the mutated one and was partially effective in distinguishing different types of mutation. Moreover, HRM data elaboration based on a Principal Component Analysis (PCA) allowed one to group populations of lines which originated from the same transformation event. Our study demonstrates the reliability of HRM as a fast and cost-effective diagnostic tool for the screening of edited lines and the evaluation of off-target events. Full article

A central challenge in functional genomics is understanding the difference between correlative transcriptomic observations and definitive causal understanding of gene function in vivo. Poultry skeletal muscle, a system of significant agricultural and biological importance, demonstrates this challenge. While transcriptomic studies have cataloged extensive RNA expression dynamics during muscle development and in growth-related myopathies like wooden breast, establishing causative roles for these molecules is lacking. This review synthesizes how advanced genetic tools are now enabling a shift from correlation to causation in avian muscle biology. We detail how viral vectors (e.g., adenovirus, lentivirus, and RCAS) and CRISPR/Cas9 systems have provided direct in vivo validation of the functional roles of specific mRNAs, miRNAs, lncRNAs, and circRNAs in regulating myogenesis, hypertrophy, and atrophy. We contrast this success in fundamental biology with the study of myopathies, which remains largely descriptive. Here, a wealth of transcriptomic data has identified dysregulated pathways, including ECM remodeling, metabolism, and inflammation, but functional validation for most candidates is absent. We argue that the critical next step is to apply this established functional genomics toolkit to disease models. By defining causal mechanisms, this research will not only address a major agricultural issue but also provide a model for using genetic tools to dissect complex traits in a post-genomic era. Full article

Antimicrobial resistance (AMR) is a present, pressing global public health crisis associated with rising morbidity and mortality rates due to previously curable infectious disease. Targeted drug delivery is an important approach to address AMR due to its ability to improve the therapeutic performance of antibiotics without leading to any adverse effects or organ toxicities. In this review we explore molecular mechanisms of AMR and drawbacks of conventional antibiotic therapies and discuss unique drug delivery approaches to compensate these. Nanoparticulate carrier systems, stimuli-responsive systems, antibody–drug conjugates, and CRISPR-Cas systems are some of the carrier method designs that are promising for tackling hard to treat infections related to pathogenic strains and biofilms due to their features. Many of these are among the most significant advances in the field. However, there are many challenges to be overcome, with biological limitations, scaling and regulatory challenges, etc., before they can be employed in commercial applications. Materials are being developed, and an approach standardized and applicable to future work is in development to improve the efficiency of targeted delivery systems. Controlled drug delivery, which could be the answer to an increasing AMR problem, will not only help in alerting awareness among individuals but will also help in prolonging the activity of antibiotics by providing synergistic interdisciplinary solutions. This review emphasizes the complementary role of targeted drug delivery in transitioning from laboratory investigations to clinical therapy. It addresses underrepresented aspects, including new materials, scalability, regulatory considerations, and ethical implications, while offering a roadmap for translating innovations into next-generation antimicrobials. Full article

The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications. Full article

Amyloid cardiomyopathy (ACM), driven by transthyretin (TTR) and immunoglobulin light chain (LC) amyloid fibrils, remains a major clinical challenge due to limited mechanistic understanding and insufficient preclinical models. Human-induced pluripotent stem cells (iPSCs) have emerged as a transformative platform to model ACM, offering patient-specific and genetically controlled systems. In this review, we summarize recent advances in the use of iPSC-derived cardiomyocytes (iPSC-CMs) in both two-dimensional (2D) monolayer cultures and three-dimensional (3D) constructs—including spheroids, organoids, cardiac microtissues, and engineered heart tissues (EHTs)—for disease modeling, mechanistic research, and drug discovery. While 2D culture of iPSC-CMs reproduces hallmark proteotoxic phenotypes such as sarcomeric disorganization, oxidative stress, and apoptosis in ACM, 3D models provide enhanced physiological relevance through incorporating multicellularity, extracellular matrix interactions, and mechanical load-related features. Genome editing with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 further broadens the scope of iPSC-based models, enabling isogenic comparisons and the dissection of mutation-specific effects, particularly in transthyretin-related amyloidosis (ATTR). Despite limitations such as cellular immaturity and challenges in recapitulating aging-associated phenotypes, ongoing refinements in differentiation, maturation, and dynamic training of iPSC-cardiac models hold great promise for overcoming these barriers. Together, these advances position iPSC-based systems as powerful human-relevant platforms for modeling and elucidating disease mechanisms and accelerating therapeutic development to prevent ACM. Full article

Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5′-SNAAAG-3′, and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems. Full article

White Spot Syndrome Virus (WSSV) is one of the most devastating viral pathogens affecting shrimp, causing severe economic losses to the global farmed shrimp trade. The globalization of live shrimp trade and waterborne transmission have facilitated the rapid spread of WSSV across major shrimp-producing countries since its initial emergence. The present review gives an updated account of WSSV biology, pathology, transmission dynamics, and recent developments in control measures. The virus, a double-stranded DNA virus of the Nimaviridae family, utilizes advanced immune evasion strategies, resulting in severe mortality. Shrimp lack adaptive immunity and hence rely predominantly on innate immunity, which is insufficient to mount an effective response against severe infections. Traditional disease control measures such as augmented biosecurity, selective breeding, and immunostimulants have, despite extensive research, achieved only limited success. New biotechnological tools such as RNA interference, CRISPR-Cas gene editing, and nanotechnology offer tremendous potential for disease mitigation. In parallel, the development of DNA and RNA vaccines targeting WSSV structural proteins, such as VP28, holds significant promise for stimulating the shrimp immune system. This review highlights the urgent need for a convergent approach to sustainable disease management in global shrimp aquaculture, with interdisciplinarity playing a pivotal role in shaping the future of WSSV control. Full article

In an era of global warming, sustainable agriculture, which emphasises the conservation of biodiversity and the rational use of natural resources, is growing in importance. One of the key elements is to increase the genetic diversity of crops through the use of crop wild relatives (CWRs) and local varieties, which provide a source of genes for resistance to biotic and abiotic stresses. Modern agricultural systems are characterised by low biodiversity, which increases the susceptibility of plants to diseases and pests. Growing mixtures of varieties, both intra- and interspecific, is a practical strategy to increase plant resistance, stabilise yields and reduce pathogen pressure. This manuscript has a review character and synthesises the current literature on the use of CWRs, local varieties, and variety mixtures in sustainable agriculture. The main research question of the study is to what extent plant genetic resources, including CWRs and local varieties, as well as the cultivation of variety mixtures, can promote plant resistance, stabilise yields and contribute to sustainable agriculture under climate change. The objectives of the study are to assess the role of genetic resources and variety mixtures in maintaining biodiversity and yield stability, and to analyse the potential of CWRs and local varieties in enhancing plant resistance. Additionally, the study investigates the impact of variety mixtures in reducing disease and pest development, and identifies barriers to the use of genetic resources in breeding along with strategies to overcome them. The study takes an interdisciplinary approach including literature and gene bank data analysis (in situ and ex situ), field trials of cultivar mixtures under different environmental conditions, genetic and molecular analysis of CWRs, the use of modern genome editing techniques (CRISPR/Cas9) and assessment of ecological mechanisms of mixed crops such as barrier effect, and induced resistance and complementarity. In addition, the study considers collaboration with participatory and evolutionary breeding programmes (EPBs/PPBs) to adapt local varieties to specific environmental conditions. The results of the study indicate that the integration of plant genetic resources with the practice of cultivating variety mixtures creates a synergistic model that enhances plant resilience and stabilises yields. This approach also promotes agroecosystem conservation, contributing to sustainable agriculture under climate change. Full article

Microfluidics enables precise control of fluid movement within microchannels, facilitating the generation of microdroplets at high frequencies. This technology provides a unique platform for conducting biological and chemical experiments, enhancing throughput and sensitivity, particularly in single-cell analysis. The microdroplet environment enhances interactions between cells and gene delivery materials, resulting in greater contact area, higher reagent concentration, and improved diffusion for both eukaryotic and prokaryotic cells. This review discusses the advantages and limitations of transfection and transformation within microdroplet technologies, highlighting their potential to improve gene editing efficiency while addressing challenges related to delivery mechanisms and cellular uptake rates. The integration of microdroplet technology with advanced gene editing tools, such as CRISPR/Cas9, promises to streamline processes and improve outcomes in various applications, including therapeutic interventions, vaccine development, regenerative medicine, and personalized medicine. These advancements could lead to more precise targeting of genetic modifications, resulting in tailored therapies that better meet individual patient needs. Overall, the integration of gene delivery in microdroplets represents a significant leap in biotechnology, enhancing the efficacy of gene delivery systems and opening new avenues for research and development in precision medicine. Full article

Genome editing technologies, including CRISPR/Cas9, TALENs, and ZFNs, offer promising approaches to disrupt HPV oncogenes E6 and E7, thereby restoring tumor-suppressor pathways. In this review, we summarize recent preclinical findings demonstrating selective apoptosis and tumor regression in HPV-positive cell and animal models, as well as early-phase clinical studies exploring local CRISPR-based therapies. We also compare the relative strengths and limitations of major editing platforms, discuss delivery strategies, and highlight their potential integration with immunotherapy and conventional treatments. While preclinical studies show encouraging efficacy (e.g., up to 60% tumor regression in xenograft models and marked reactivation of p53/pRb pathways), translation into routine practice remains limited by challenges such as efficient delivery, minimizing off-target effects, long-term safety, cost, and ethical considerations. Continued optimization of high-fidelity nucleases, tissue-specific delivery systems, and genotype-tailored guide RNAs will be essential. Genome editing therefore represents a potential future addition to the therapeutic landscape of HPV-related diseases, but substantial barriers must be addressed before clinical implementation. Full article