Search Results (167)

Cyanobacteria harbor sophisticated molecular defense systems that have evolved over billions of years to protect against viral invasion and foreign genetic elements. These ancient photosynthetic organisms possess a diverse array of restriction-modification (R-M) systems and CRISPR-Cas arrays that present challenges for genetic engineering, but also offer unique opportunities for cancer-targeted biotechnological applications. These systems exist in prokaryotes mainly as defense mechanisms but they are currently used in molecular applications as gene editing tools. Moreover, latest developments in nucleases such as zinc finger nucleases (ZFNs), TALENs (transcription-activator-like effector nucleases) are discussed. A comprehensive genomic analysis of 126 cyanobacterial species found 89% encode multiple R-M systems, averaging 3.2 systems per genome, creating formidable barriers to transformation but also providing molecular machinery that could be harnessed for precise recognition and targeting of cancer cells. This review critically examines the dual nature of these defense systems, their ecological functions, and the emerging strategies to translate their molecular precision into advanced anticancer therapeutics. Hence, the review main objectives are to explore the recent understanding of these mechanisms and to exploit the knowledge gained in opening new avenues for cancer-focused targeted interventions, while acknowledging the significant challenges to translate these systems from laboratory curiosities to practical applications. Full article

Thrips are cosmopolitan agricultural pests and important vectors of plant viruses, and the increasing coexistence of multiple morphologically similar species has intensified the demand for species-specific molecular identification. However, traditional morphological identification and PCR assays using universal primers are often inadequate for mixed-species samples and field-adaptable application. In this study, we developed a species-specific molecular identification framework targeting a polymorphism-rich region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which is more time-efficient than sequencing-based COI DNA barcoding, for four economically important thrips species in southern China, including the globally invasive Frankliniella occidentalis. By aligning COI sequences, polymorphism-rich regions were identified and used to design four species-specific primer pairs, each containing a diagnostic 3′-terminal nucleotide. These primers were combined with a PBS-based DNA extraction workflow optimized for single-insect samples that minimizes dependence on column-based purification. The assay achieved a practical detection limit of 1 ng per reaction, demonstrated species-specific amplification, and maintained reproducible amplification at DNA inputs of ≥1 ng per reaction. Notably, PCR inhibition caused by crude extracts was effectively alleviated by fivefold dilution. Although the chemical identities of the inhibitors remain unknown, interspecific variation in inhibition strength was observed, with T. hawaiiensis exhibiting the strongest suppression, possibly due to differences in lysate composition. This integrated framework balances target specificity, operational simplicity, and dilution-mitigated inhibition, providing a field-adaptable tool for thrips species identification and invasive species monitoring. Moreover, it provides a species-specific molecular foundation for downstream integration with visual nucleic acid detection platforms, such as the CRISPR/Cas12a system, thereby facilitating the future development of portable molecular identification workflows for small agricultural pests. Full article

The cornea is an avascular, immune-privileged tissue critical to maintaining transparency, optimal light refraction, and protection from microbial and immunogenic insults. Corneal neovascularization (CoNV) is a pathological sequela of multiple anterior segment diseases and presents a major cause for reduced visual acuity and overall quality of life. Various aetiologies, including infection (e.g., herpes simplex), inflammation (e.g., infective keratitis), hypoxia (e.g., contact lens overuse), degeneration (e.g., chemical burns), and trauma, disrupt the homeostatic avascular microenvironment, triggering an overactive compensatory response. This response is governed by a complex interplay of pro- and anti-angiogenic factors. This review investigates the potential for these mediators to serve as therapeutic targets. Current therapeutic strategies for CoNV encompass topical corticosteroids, anti-VEGF injections, fine-needle diathermy, and laser modalities including argon, photodynamic therapy and Nd:YAG. Emerging therapies involve steroid-sparing immunosuppressants (including cyclosporine and rapamycin), anti-fibrotic agents and advanced drug delivery systems, including ocular nanosystems and viral vectors, to enhance drug bioavailability. Adjunctive therapy to attenuate the protective corneal epithelium prior to target neovascular plexi are further explored. Gene-based approaches, such as Aganirsen (antisense oligonucleotides) and CRISPR/Cas9-mediated VEGF-A editing, have shown promise in preclinical studies for CoNV regression and remission. Given the multifactorial pathophysiology of CoNV, combination therapies targeting multiple molecular pathways may offer improved visual outcomes. Case studies of CoNV highlight the need for multifaceted approaches tailored to patient demographics and underlying ocular diseases. Future research and clinical trials are essential to elucidate optimal therapeutic strategies and explore combination therapies to ensure better management, improved treatment outcomes, and long-term remission of this visually disabling condition. Full article

The CRISPR–Cas9 system has significantly advanced genome editing but remains constrained by its requirement for specific protospacer adjacent motifs (PAMs). To overcome this limitation, PAM-relaxed nucleases, including the novel near-PAMless chimeric SpRYc, have been developed. Here, we evaluated SpRYc editing activity across multiple experimental systems, including human HEK293 and CEM-R5 cells, as well as Drosophila melanogaster S2 cells and embryos. In HEK293 cells, SpRYc exhibited broad PAM compatibility, enabling editing at non-canonical PAMs, albeit with reduced and variable efficiency at canonical NGG sites compared to SpCas9. This context dependency was more pronounced in CEM-R5 T cells, where SpRYc activity at endogenous CXCR4 and B2M loci was largely restricted to NGG PAMs. In contrast, unlike SpCas9, SpRYc displayed negligible genome-editing activity in Drosophila embryos in vivo. Notably, the transcriptional activator dSpRYc-VPR showed robust activity in Drosophila S2 cells at both canonical and non-canonical PAMs. Reduced chromatin occupancy of dSpRYc-VPR suggests a balance between expanded PAM recognition and DNA-binding stability, providing a mechanistic explanation for context-dependent performance of SpRYc. Overall, our results highlight that expanded targeting flexibility comes at the cost of variable efficiency, underscoring the need for extensive locus- and context-specific validation of PAM-relaxed genome-editing tools. Full article

Bulleidia extructa strain PP_925, isolated from the periodontal pocket of a patient with periodontitis, is a Gram-positive Bacillota with an unusually compact genome of 1.38 Mb. Phylogenomic analyses place PP_925 within Erysipelotrichales and show close relatedness of Bulleidia to Solobacterium and Lactimicrobium, as well as the existence of previously undescribed related clades. The metabolic repertoire of PP_925 is strongly reduced: it retains glycolysis, the phosphotransacetylase–acetate kinase pathway, and arginine catabolism but lacks the tricarboxylic acid cycle and most de novo biosynthetic pathways for amino acids, nucleotides, fatty acids, cofactors, and vitamins, implying reliance on salvage and cross-feeding. Phylogenetic inference indicates independent peptidoglycan losses in multiple mycoplasma Erysipelotrichia-related lineages, while PP_925 has retained an ancestral Gram-positive cell wall despite extensive genomic reduction. The genome preserves systems crucial for host interaction and adaptability, including a horizontally acquired tad locus encoding type IV pili, a comG competence system, and several adherence-associated virulence factors. Defense mechanisms are diverse and include a CRISPR-Cas II-A system, a type II restriction–modification module adjacent to Gao_Qat-like genes, and the Wadjet system in a genome without prophages; CRISPR spacers indicate repeated encounters with Bacillota phages. Comparative genomics of PP_925 and related strains reveals a small core genome with lineage-specific adhesion and defense modules, indicating recent shared ancestry combined with adaptive flexibility under substantial genome reduction. Full article

Mesenchymal stem cells are multipotent stromal cells with immunomodulatory, anti-inflammatory, and trophic properties that support tissue repair and regeneration. Increasing evidence suggests that their therapeutic effects are primarily mediated by paracrine signaling, especially through extracellular vesicles, which can cross the blood–brain barrier and act as cell-free therapeutic agents. Preclinical and clinical studies in stroke, multiple sclerosis, spinal cord injury, and neurodegenerative diseases report encouraging outcomes but also reveal major challenges, including limited engraftment, donor-related heterogeneity, incomplete understanding of mechanisms, and potential oncogenic risks. Recent advances in biotechnology—such as mesenchymal stem cell-derived extracellular vesicles, genetic engineering using CRISPR/Cas9 or viral vectors, 3D culture systems, and bioengineered delivery platforms—offer new opportunities to overcome these limitations. Early clinical trials demonstrate promising safety and functional improvements, yet results remain inconsistent, highlighting the need for standardized protocols and large-scale controlled studies. This review outlines current knowledge, key challenges, and emerging strategies aimed at optimizing mesenchymal stem cell-based approaches for regenerative neurology. Full article

Low-temperature (LT) stresses (cold and frost) are major abiotic factors limiting plant growth and productivity. LT induces numerous physiological and biochemical changes in plants, changes hormonal balance and photosynthetic efficiency. Stress induced by LT often leads to yield losses in crops. While plants like maize and cucumber are highly sensitive to cold, winter cereals such as wheat and rye suffer mainly from severe frosts. Ongoing climate change and temperature fluctuations further increase the risk of LT-induced damage. To counteract the problems connected with LT stress, multiple strategies have been developed to enhance plant tolerance. Agrotechnical practices and biochemical treatments involving the application of phytohormones or osmoprotectants are designed to improve plant tolerance to LT. Beneficial plant–microbe interactions also contribute to alleviating LT stress. In addition, genetic engineering offers powerful tools for creating new cultivars that are more tolerant to LT. The CRISPR/Cas system, in particular, enables precise modifications and represents a promising tool for advancing sustainable agriculture. Integrated methods of protection are crucial for securing food supplies, especially under conditions of a changing climate. This mini-review summarises strategies for protecting plants against LT stress, with special attention paid to crop plants. Full article

African swine fever virus (ASFV) is a high-consequence pathogen that causes African swine fever (ASF), for which mortality rates can reach 90–100%, with death typically occurring within 14 days. ASF is currently a highly contagious pandemic disease responsible for extensive losses in pig production in multiple affected countries suffering from extended outbreaks. While a limited number of vaccines to prevent ASF are in use in south-east Asia, vaccines are not widely available, are only effective against highly homologous strains of ASFV, and must be used prior to an outbreak on a farm. Currently, there is no treatment for ASF and culling affected farms is the only response to outbreaks on farms to try and prevent spreading. CRISPR/Cas systems evolved as an adaptive immune response in bacteria and archaea that function by cleaving and disrupting the genomes of invading bacteriophage pathogens. CRISPR technology has since been leveraged into an array of endonuclease-based systems used for nucleic acid detection, targeting, genomic cleavage, and gene editing, making them particularly well-suited for development as sequence-specific therapeutic modalities. The programmability of CRISPR-based therapeutics offers a compelling new way to rapidly and specifically target pathogenic viral genomes simply by using different targeting guide RNAs (gRNA) as an adaptable antiviral modality. Here, we demonstrate for the first time a specific CRISPR/Cas9 multiplexed gRNA system that targets the African swine fever viral genome, resulting in sequence-specific cleavage, leading to the reduction in the viral load in infected animals, and subsequent recovery from an otherwise lethal dose of ASFV. Moreover, animals that recovered had protective immunity to subsequent homologous ASFV infection. Full article

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

Canopy FGF signaling regulator 3 (CNPY3) is a cochaperone of the molecular chaperone GRP94. CNPY3 is critical for the post-translational maturation of toll-like receptors and for regulating inflammasome signaling. However, the role of CNPY3 in cancer development and progression is still not fully understood. In this study, we aimed to investigate the role of CNPY3 in human breast cancer progression and metastasis. We used genomic and clinical information from multiple databases to profile CNPY3 and GRP94 in human cancers. We found that CNPY3 and GRP94 were elevated in human breast cancers compared to normal tissue. Higher expression of CNPY3 correlated with cancer progression and poor clinical outcomes in breast cancers. We confirmed these findings using a human breast cancer tissue array. We silenced CNPY3 in human breast cancer cells using a CRISPR/Cas9 system. For the first time, we found that deletion of CNPY3 significantly reduced tumor growth and metastasis in vitro and in vivo. Additionally, network and enrichment analyses revealed that changes in the unfolded protein response pathway and immune-related genes were significantly dependent on alterations in CNPY3 and GRP94. This study suggests that CNPY3 is a potential biomarker and novel therapeutic target for cancers. 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

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

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) nucleases enable precise genome editing, but off-target cleavage remains a critical challenge. Here, we report the development of MAD7_HF, a high-fidelity variant of the MAD7 nuclease engineered through a bacterial screening system leveraging the DNA gyrase-targeting toxic gene ccdB. This system couples survival to efficient on-target cleavage and minimal off-target activity, mimicking the transient action required for high-precision editing. Through iterative selection and sequencing validation, we identified MAD7_HF, harboring three substitutions (R187C, S350T, K1019N) that enhanced discrimination between on- and off-target sites. In Escherichia coli assays, MAD7_HF exhibited a >20-fold reduction in off-target cleavage across multiple mismatch contexts while maintaining on-target efficiency comparable to wild-type MAD7. Structural modeling revealed that these mutations stabilize the guide RNA-DNA hybrid at on-target sites and weaken interactions with mismatched sequences. This work establishes a high-throughput bacterial screening strategy that allows the identification of Cas12a variants with improved specificity at a given target site, providing a useful framework for future efforts to develop precision genome-editing tools. Full article

Plants have evolved a complex, multilayered immune system that integrates molecular recognition, signaling pathways, epigenetic regulation, and small RNA-mediated control. Recent studies have shown that DNA-level regulatory mechanisms, such as RNA-directed DNA methylation (RdDM), histone modifications, and chromatin remodeling, are critical for modulating immune gene expression, allowing for rapid and accurate pathogen-defense responses. The epigenetic landscape not only maintains immunological homeostasis but also promotes stress-responsive transcription via stable chromatin modifications. These changes contribute to immunological priming, a process in which earlier exposure to pathogens or abiotic stress causes a heightened state of preparedness for future encounters. Small RNAs, including siRNAs, miRNAs, and phasiRNAs, are essential for gene silencing before and after transcription, fine-tuning immune responses, and inhibiting negative regulators. These RNA molecules interact closely with chromatin features, influencing histone acetylation/methylation (e.g., H3K4me3, H3K27me3) and guiding DNA methylation patterns. Epigenetically encoded immune memory can be stable across multiple generations, resulting in the transgenerational inheritance of stress resilience. Such memory effects have been observed in rice, tomato, maize, and Arabidopsis. This review summarizes new findings on short RNA biology, chromatin-level immunological control, and epigenetic memory in plant defense. Emerging technologies, such as ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing), ChIP-seq (Chromatin Immunoprecipitation followed by Sequencing), bisulfite sequencing, and CRISPR/dCas9-based epigenome editing, are helping researchers comprehend these pathways. These developments hold an opportunity for establishing epigenetic breeding strategies that target the production of non-GMO, stress-resistant crops for sustainable agriculture. Full article

The rapid evolution of CRISPR technology has revolutionized molecular biology, and among the various systems, CRISPR/Cas12a stands out for its high specificity and efficient collateral cleavage activity. This review article focuses on the recent advancements and applications of split technology within the CRISPR/Cas12a framework, highlighting its transformative role in molecular diagnostics and biosensing. Split technology innovatively divides functional nucleic acid components into modular segments that are activated by specific targets, significantly enhancing the specificity and sensitivity of biosensors. This design addresses the inherent limitations of traditional sensor systems, enabling the direct detection of ultrashort nucleic acids and improved discrimination of single-nucleotide variants, thereby facilitating the simultaneous detection of multiple biomolecules. The versatility of split-enabled biosensors extends beyond genetic testing, making them valuable tools in diagnostics, therapeutics, and environmental science. Despite challenges such as crRNA degradation and reassembly kinetics, ongoing research and engineering solutions continue to enhance the stability and performance of these systems. This review synthesizes the foundational principles, recent advancements, and potential applications of split technology while also identifying challenges and opportunities for future exploration. Ultimately, our insights provide a comprehensive resource to leverage the full potential of CRISPR/Cas12a-based split technology in advancing biosensing methodologies and clinical applications. Full article