Search Results (1,401)

Background/Objectives: Lyme disease diagnosis remains challenging due to the limitations of current methods. While PCR-based assays are widely used, their sensitivity can be affected by sample type and the inhibition of host DNA. This study aimed to evaluate the feasibility and sensitivity of a CRISPR/Cas12-based detection system for Borrelia burgdorferi sensu lato, comparing its performance with real-time PCR. Methods: DNA from three Borrelia genospecies (B. burgdorferi, B. garinii, and B. afzelii) was amplified targeting the OspA gene. Detection was performed using a Cas12/crRNA system with a fluorescent ssDNA reporter. Sensitivity assays were conducted on serial dilutions of Borrelia DNA, with and without human genomic DNA, and results were compared with qPCR. Results: Direct detection of Borrelia DNA without amplification was not feasible. However, when combined with PCR, the Cas12/crRNA system reliably detected as few as 5 genome copies per reaction. End-point PCR extended to 60 cycles improved detection robustness for B. garinii and B. afzelii, although sensitivity decreased in the presence of human genomic DNA. Conclusions: The Cas12/crRNA-based system offers a sensitive and accessible alternative to qPCR, especially in settings lacking real-time PCR instrumentation. Future developments may include integration with isothermal amplification and microfluidic platforms to enhance direct detection capabilities. Full article

Background/Objectives: Liquid biopsy using cfDNA has emerged as a promising, minimally invasive alternative to traditional tissue biopsy for detecting cancer-associated mutations. However, the extremely low proportion of mutant DNA in cfDNA poses a major challenge for accurate detection, especially when using conventional sequencing methods. To address this limitation, we sought to develop a highly sensitive diagnostic strategy to selectively enrich rare mutant sequences and improve the detection of clinically important mutations in patients with NSCLC. Methods: We established a CRISPR/Cas12a-based diagnostic system designed to selectively cleave WT DNA, thereby increasing the relative abundance of mutant DNA in cfDNA samples. Following Cas12a-mediated WT cleavage, the remaining DNA was subjected to PCR amplification for mutation identification. The system was applied to plasma cfDNA from blood samples of 48 NSCLC patients to evaluate its ability to detect two major EGFR mutations: L858R and exon 19 deletion. Results: The CRISPR/Cas12a-based diagnostic system effectively identified low-frequency EGFR mutations in cfDNA. Specifically, all 7 L858R-positive samples and 6 out of 11 samples harboring exon 19 deletions—previously validated through tissue biopsy—were successfully detected. This demonstrated a high degree of concordance between our liquid biopsy approach and conventional diagnostic methods. Conclusions: Our findings highlight the potential of the CRISPR/Cas12a-based mutation enrichment system as a powerful tool for detecting rare oncogenic mutations in liquid biopsy samples. This technique enhances diagnostic sensitivity and could be broadly applicable for the non-invasive detection of various genetic alterations in cancer and other diseases. Full article

Avian reovirus (ARV), a highly pathogenic agent in poultry, causes severe economic losses through immunosuppression and secondary infections. Traditional diagnostic methods like reverse transcription quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) face limitations in resource-limited settings due to equipment dependency and prolonged processing. To address this, we developed a rapid, portable detection method integrating reverse transcription–recombinase-aided amplification (RT-RAA) with CRISPR/Cas12a. By targeting the conserved P17-coding region of the ARV S1 gene, this assay amplifies viral RNA isothermally (37 °C) within 20 min, followed by Cas12a-mediated collateral cleavage of fluorescent or lateral flow reporters for visual readout. The method achieved a sensitivity of 1 copy/μL, surpassing RT-qPCR (10 copies/μL), and completed detection in 40 min. Specificity tests against non-target pathogens confirmed zero cross-reactivity. Utilizing a portable incubator and low-cost visual tools, this platform eliminates reliance on thermocyclers and skilled personnel. Its field-deployable design enables on-site diagnosis, facilitating early ARV detection to mitigate outbreaks and economic losses in poultry farming. This study provides a paradigm shift in avian pathogen surveillance, combining speed, sensitivity, and accessibility for global agricultural and public health applications. Full article

Developing climate-resilient and high-quality cotton cultivars remains an urgent challenge, as the key target traits yield, fibre properties, and stress tolerance are highly polygenic and strongly influenced by genotype–environment interactions. Recent advances in chromosome-scale genome assemblies, pan-genomics, and haplotype-resolved resequencing have greatly enhanced the capacity to identify causal variants and recover non-reference alleles linked to fibre development and environmental adaptation. Parallel progress in functional genomics and precision genome editing, particularly CRISPR/Cas, base editing, and prime editing, now enables rapid, heritable modification of candidate loci across the complex tetraploid cotton genome. When integrated with high-throughput phenotyping, genomic selection, and machine learning, these approaches support predictive ideotype design rather than empirical, trial-and-error breeding. Emerging digital agriculture tools, such as digital twins that combine genomic, phenomic, and environmental data layers, allow simulation of ideotype performance and optimisation of trait combinations in silico before field validation. Speed breeding and phenomic selection further shorten generation time and increase selection intensity, bridging the gap between laboratory discovery and field deployment. However, the large-scale implementation of these technologies faces several practical constraints, including high infrastructural costs, limited accessibility for resource-constrained breeding programmes in developing regions, and uneven regulatory acceptance of genome-edited crops. However, reliance on highly targeted genome editing may inadvertently narrow allelic diversity, underscoring the need to integrate these tools with broad germplasm resources and pangenomic insights to sustain long-term adaptability. To realise these opportunities at scale, standardised data frameworks, interoperable phenotyping systems, robust multi-omic integration, and globally harmonised, science-based regulatory pathways are essential. This review synthesises recent progress, highlights case studies in fibre, oil, and stress-resilience engineering, and outlines a roadmap for translating integrative genomics into climate-smart, high-yield cotton breeding programmes. Full article

Exosomes, naturally derived extracellular vesicles, have emerged as powerful bio-nanocarriers in precision medicine. Their endogenous origin, biocompatibility, and ability to encapsulate and deliver diverse therapeutic payloads position them as transformative tools in drug delivery, gene therapy, and regenerative medicine. This review presents a comprehensive analysis of exosome-based therapeutics across multiple biomedical domains, including cancer, neurological and infectious diseases, immune modulation, and tissue repair. Exosomes derived from stem cells, immune cells, or engineered lines can be loaded with small molecules, RNA, or CRISPR-Cas systems, offering highly specific and low-immunogenic alternatives to viral vectors or synthetic nanoparticles. We explore endogenous and exogenous loading strategies, surface functionalization techniques for targeted delivery, and innovations that allow exosomes to traverse physiological barriers such as the blood–brain barrier. Furthermore, exosomes demonstrate immunomodulatory and regenerative properties in autoimmune and degenerative conditions, with promising roles in skin rejuvenation and cosmeceuticals. Despite their potential, challenges remain in large-scale production, cargo loading efficiency, and regulatory translation. Recent clinical trials and industry efforts underscore the accelerating momentum in this field. Exosomes represent a promising platform in precision medicine, though further standardization and validation are required before widespread clinical use. This review offers critical insights into current technologies, therapeutic mechanisms, and future directions to unlock the full translational potential of exosomes in clinical practice. Full article

N6-methyladenosine (m⁶A) modifications are among the most prevalent epigenetic marks in eukaryotic RNAs, regulating both coding and non-coding RNAs and playing a pivotal role in RNA metabolism. Given their widespread influence, m⁶A modifications are deeply implicated in the pathogenesis of various cancers, including highly aggressive malignancies such as lung cancer, melanoma, and liver cancer. Dysregulation of m⁶A dynamics—marked by an imbalance in methylation and demethylation—can drive tumor progression, enhance metastatic potential, increase aggressiveness, and promote drug resistance, while also exerting context-dependent tumor-suppressive effects. Given this dual role, precise modulation of m⁶A levels and the activity of its regulatory enzymes (writers, erasers, and readers) represent a promising therapeutic avenue. In this review, we highlight recent advances in targeting m⁶A machinery, including small-molecule inhibitors, antisense oligonucleotides, and CRISPR/Cas-based editing tools, capable of both writing and erasing m⁶A marks and altering m⁶A methylation sites per se. By evaluating these strategies, we aim to identify the most effective approaches for restoring physiological m⁶A homeostasis or for strategically manipulating the m⁶A machinery for therapeutic benefit. Full article

Recent technological breakthroughs have enabled multidimensional phenotyping, with unprecedented single-cell resolution and genome-wide coverage, across multiple osteoarthritis (OA)-relevant tissues, such as articular cartilage, synovium, infrapatellar fat pad, and subchondral bone. The majority of the single nucleotide variations (SNVs) that have been associated with OA are located in non-protein coding regions and confer risk for disease by altering the expression level, instead of the amino acid sequence of the gene product. These data have shaped the concept of OA as a polygenic disease, where genetic factors disrupt the chromatin landscape in disease-relevant cells, leading to aberrant expression of effector genes. Pharmacologic manipulation of the OA-driving epigenetic landscape has recently emerged as an attractive path for the development of disease-modifying drugs. Novel clustered regulatory interspaced short palindromic repeats (CRISPR)-based technologies provide opportunities for precise epigenetic editing at the desired genomic regions and may allow a targeted transcriptional regulation of disease-relevant genes in disease-relevant cells. The aim of the present narrative review is to summarize the emerging data on the role of epigenetic factors and chromatin structure as calibrators of the risk for developing OA and to discuss the opportunities and challenges arising from the use of chromatin landscape to guide drug discovery. Full article

β-thalassemia is an inherited blood disorder caused by mutations in the β-globin (HBB) gene, leading to reduced or absent β-globin production, resulting in chronic anemia. While current therapies, including blood transfusions and hematopoietic stem cell transplantation, offer symptomatic relief, they are limited by complications and their limited accessibility. CRISPR-based gene editing technologies provide new therapeutic avenues by enabling the precise correction of HBB mutations or the reactivation of fetal hemoglobin (HbF) through the targeting of regulatory elements such as BCL11A. These approaches have shown promising preclinical and clinical outcomes. However, efficient and safe delivery remains a major challenge. Viral vectors offer high efficiency but raise concerns about immunogenicity and insertional mutagenesis, whereas non-viral systems such as lipid nanoparticles and engineered exosomes offer lower toxicity and modularity but face targeting limitations. This review highlights recent progress in CRISPR-based therapies for β-thalassemia and emerging delivery strategies to enhance clinical translation. Full article

Worldwide, polymicrobial infections (PMIs) account for an estimated 20–50% of severe clinical infection cases, with biofilm-associated and device-related infections reaching 60–80% in hospitalized patients. This review discusses the clinical burden of major infections in which PMIs are almost inevitable, such as diabetic foot infections, intra-abdominal infections, pneumonia, and biofilm-associated device infections. Globally, the PMI landscape is diverse; however, the Indian subcontinent is a PMI hotspot where high comorbidities, endemic antimicrobial resistance, and underdeveloped diagnostic capacity elevate the risks of poor outcomes. Existing diagnostic like culture-based methods, PCR panels, sequencing, and biomarker-based assays are constrained by sensitivity, turnaround times (TATs), and high costs. Vulnerable populations, particularly neonates, the elderly, immunocompromised patients, and socioeconomically marginalized groups, show case-fatality rates 2-fold higher than monomicrobial infections in similar settings. Emerging diagnostic solutions include CRISPR-based multiplex assays, artificial intelligence-based metagenomic platforms, and sensitive biosensors with point-of-care applicability. These technologies show potential in reducing the TAT (<2 h) with high accuracy (>95%). However, their translation to real-world settings depends critically on affordability, integration into healthcare pathways, and supportive policy. This will provide equitable diagnostic access, particularly in low- and middle-income countries (LMICs). Full article

Spinal cord injury (SCI) is a neurological condition often resulting in permanent motor and sensory deficits, for which effective treatments remain limited. RNA interference (RNAi) is a post-transcriptional mechanism of the downregulation of gene expression mediated by small interfering RNAs. RNAi has demonstrated therapeutic efficacy in various neurological disorders, positioning it as a promising yet underexplored therapeutic strategy for SCI. Here, we provide a focused overview of the key pathological processes in SCI, including primary mechanical injury and secondary cascades such as inflammation, mitochondrial dysfunction, excitotoxicity, oxidative stress, multiple forms of cell death, and others. The potential of RNAi to selectively silence genes implicated in these pathological processes, thereby enhancing neuroprotection and functional recovery, is highlighted. We point out that not only protein-coding genes, but non-coding RNAs (ncRNAs) are suitable targets for RNAi. Novel RNAi tools such as CRISPR-Cas13 might revolutionize the field and offer new opportunities for SCI therapy. However, despite all these promising findings, relevant translational studies of RNAi remain scarce. Challenges related to delivery methods, long-term efficacy, and cell-specific targeting must be addressed. Importantly, combining RNAi with other strategies such as cell- or biomaterial-based therapies may enhance therapeutic outcomes. Future investigations should prioritize systematic comparisons of RNAi targets and delivery systems, ideally at single-cell resolution and in different SCI models, to identify the most relevant molecular pathways for clinical translation. Overall, RNAi represents a compelling but still underdeveloped approach for SCI therapy, requiring continued refinement to reach clinical application. Full article

Organoid technology has emerged as a revolutionary tool in cancer research, offering physiologically accurate, three-dimensional models that preserve the histoarchitecture, genetic stability, and phenotypic complexity of primary tumors. These self-organizing structures, derived from adult stem cells, induced pluripotent stem cells, or patient tumor biopsies, recapitulate critical aspects of tumor heterogeneity, clonal evolution, and microenvironmental interactions. Organoids serve as powerful systems for modeling tumor progression, assessing drug sensitivity and resistance, and guiding precision oncology strategies. Recent innovations have extended organoid capabilities beyond static culture systems. Integration with microfluidic organoid-on-chip platforms, high-throughput CRISPR-based functional genomics, and AI-driven phenotypic analytics has enhanced mechanistic insight and translational relevance. Co-culture systems incorporating immune, stromal, and endothelial components now permit dynamic modeling of tumor–host interactions, immunotherapeutic responses, and metastatic behavior. Comparative analyses with conventional platforms, 2D monolayers, spheroids, and patient-derived xenografts emphasize the superior fidelity and clinical potential of organoids. Despite these advances, several challenges remain, such as protocol variability, incomplete recapitulation of systemic physiology, and limitations in scalability, standardization, and regulatory alignment. Addressing these gaps with unified workflows, synthetic matrices, vascularized and innervated co-cultures, and GMP-compliant manufacturing will be crucial for clinical integration. Proactive engagement with regulatory frameworks and ethical guidelines will be pivotal to ensuring safe, responsible, and equitable clinical translation. With the convergence of bioengineering, multi-omics, and computational modeling, organoids are poised to become indispensable tools in next-generation oncology, driving mechanistic discovery, predictive diagnostics, and personalized therapy optimization. Full article

The persistent residual tumor cells that survive after chemotherapy are a major cause of treatment failure, but their survival mechanisms remain largely elusive. These cancer cells are typically characterized by a quiescent state with suppressed activity of MYC and MTOR. We observed that the MYC-suppressed persistent triple-negative breast cancer (TNBC) cells are metabolically flexible and can upregulate mitochondrial oxidative phosphorylation (OXPHOS) genes and respiratory function (“OXPHOS-high” cell state) in response to DNA-damaging anthracyclines such as doxorubicin, but not to taxanes. The elevated biomass and respiratory function of mitochondria in OXPHOS-high persistent cancer cells were associated with mitochondrial elongation and remodeling, suggestive of increased mitochondrial fusion. A genome-wide CRISPR editing screen in doxorubicin-persistent OXPHOS-high TNBC cells revealed the BCL-XL gene as the top survival dependency in these quiescent tumor cells, but not in their untreated proliferating counterparts. Quiescent OXPHOS-high TNBC cells were highly sensitive to BCL-XL inhibitors, but not to inhibitors of BCL2 and MCL1. Interestingly, inhibition of BCL-XL in doxorubicin-persistent OXPHOS-high TNBC cells rapidly abrogated mitochondrial elongation and respiratory function, followed by caspase 3/7 activation and cell death. The platelet-sparing proteolysis-targeted chimera (PROTAC) BCL-XL degrader DT2216 enhanced the efficacy of doxorubicin against TNBC xenografts in vivo without induction of thrombocytopenia that is often observed with the first-generation BCL-XL inhibitors, supporting the development of this combinatorial treatment strategy for eliminating dormant tumor cells that persist after treatment with anthracycline-based chemotherapy. Full article

Damping-off disease hinders rice seedling growth and reduces yield. Current control methods, such as seed or soil sterilization, rely on chemicals that cause environmental pollution and promote pathogen resistance. As a sustainable alternative, we targeted the damping-off resistance-related gene OsDGTq1 using CRISPR/Cas9. Field experiments first verified OsDGTq1’s significance in resistance. The CRISPR/Cas9 system, delivered via Agrobacterium-mediated transformation, was used to edit OsDGTq1 in rice cultivar Ilmi. Lesions from major damping-off pathogens, Rhizoctonia solani and Pythium graminicola, were observed on G₀ plants. All 37 regenerated plants contained T-DNA insertions. Among them, edits generated by sgRNA1-1, sgRNA1-2, and sgRNA1-3 resulted in the insertion of two thymine bases as target mutations. Edited lines were assigned names and evaluated for agronomic traits, seed-setting rates, and pathogen responses. Several lines with edited target genes showed distinct disease responses and altered gene expression compared to Ilmi, likely due to CRISPR/Cas9-induced sequence changes. Further studies in subsequent generations are needed to confirm the stability of these edits and their association with resistance. These results confirm that genome editing of OsDGTq1 alters resistance to damping-off. The approach demonstrates that gene-editing technology can accelerate rice breeding, offering an environmentally friendly strategy to develop resistant varieties. Such varieties can reduce chemical inputs, prevent pollution, and minimize seedling loss, ultimately enhancing food self-sufficiency and stabilizing rice supply. Full article

Bolivian hemorrhagic fever (BHF) is a zoonotic disease caused by Mammarenavirus machupoense (MACV) featuring severe neurological and hemorrhagic symptoms and a high mortality rate. BHF is usually diagnosed by serological tests or real-time polymerase chain reaction (RT-PCR); these methods are often inaccessible in endemic regions due to a lack of laboratory infrastructure, creating a demand for sensitive and rapid equipment-free alternatives. Here, we present an isothermal method for MACV nucleic acid detection based on the Cas12a-based DETECTR system combined with recombinase polymerase amplification (RPA) in a single tube: the RT-RPA/DETECTR assay. We demonstrate the possibility of using more than one primer set for the simultaneous detection of MACV genetic variants containing multiple point mutations. The method was optimized and tested using specially developed virus-like armored particles containing the target sequence. The multiplex RT-RPA/DETECTR method achieved a limit of detection of approximately 5 × 10⁴ copies/ mL (80 aM) of armored particles. The method was validated using clinical samples spiked with virus-like particles. The assay proved to be selective and reliable in detecting certain nucleotide substitutions simultaneously. Full article

Fungi play a crucial yet often unnoticed role in our lives and the health of our planet by breaking down organic matter through their diverse enzymes or eliminating environmental contamination, enhancing biomass pretreatment, and facilitating biofuel production. They offer transformative possibilities not only for improving the production of materials they naturally produce, but also for the production of non-native and even new-to-nature materials. However, despite these promising applications, the full potential of fungi remains untapped mainly due to limitations in our ability to control and optimize their complex biological systems. This review focuses on developments that address these challenges, with specific emphasis on fungal-derived rigid and flexible materials. To achieve this goal, the application of synthetic biology tools—such as programmable regulators, CRISPR-based genome editing, and combinatorial pathway optimization—in engineering fungal strains is highlighted, and how external environmental parameters can be tuned to influence material properties is discussed. This review positions filamentous fungi as promising platforms for sustainable bio-based technologies, contributing to a more sustainable future across various sectors. Full article