Search Results (163)

Gene therapy is undergoing continued clinical translation and technological development. This progress has been marked by regulatory approvals and broadened therapeutic indications across genetic, metabolic, and oncologic diseases and disorders. The field has evolved over decades from early viral-mediated gene addition to approaches capable of targeted editing, regulation, or replacement of genetic information. These systems include base and prime editors, epigenetic modulators, CRISPR-Cas, RNA therapeutics and programmable integration platforms. When paired with increasingly sophisticated viral and nonviral delivery strategies, these technologies enable greater control over tissue targeting, duration of activity, and therapeutic exposure. Recent clinical successes, including approved ex vivo CRISPR-based therapies for hemoglobinopathies, in vivo CRISPR editing for transthyretin amyloidosis, and emerging clinical applications of base and prime editing, provide growing clinical evidence for the feasibility of genetic medicines. However, technological advancement has also made platform selection increasingly complex. Therapeutic performance is determined not by editing efficiency alone, but by the interaction among genetic precision, temporal control, dosage tunability, delivery efficiency, durability, and disease-specific safety requirements. A molecularly efficient platform may still have limited therapeutic value if it cannot reach the disease-relevant cell population at sufficient and safe exposure. In this review, we examine recent technological and clinical advances in genetic medicine with particular emphasis on developments during the past approximately five years. We propose a multidimensional framework in which gene therapy platforms are evaluated according to three intrinsic properties—genetic precision, temporal control, and dosage tunability—while delivery, clinical maturity, and disease context act as major translational constraints. This framework highlights that no single platform is universally optimal; rather, successful therapeutic design depends on matching the biological characteristics of the intervention to the requirements of the disease and target tissue. Remaining challenges in extrahepatic delivery, genomic safety, immunogenicity, manufacturing, and long-term monitoring remain important determinants of broader clinical implementation. Full article

Genetic modification and genome editing have become important tools in agriculture, animal production, biotechnology, and human medicine, but their safety and societal acceptance remain subjects of debate. This review examines genetically modified (GM) and gene-edited organisms, distinguishing transgenesis from precision genome editing technologies, including CRISPR/Cas9, base editing, and prime editing. Representative applications in crops, livestock, pharmaceutical production, and xenotransplantation are discussed, together with their regulatory framework and public perception. Current scientific assessments indicate that approved GM foods are not inherently more hazardous to human health than their conventional counterparts when evaluated case by case. Potential benefits include improved nutritional quality, biofortification, disease resistance, increased agricultural efficiency, production of therapeutic proteins, and applications in animal health and medicine. Possible concerns include allergenicity, toxicity, unintended genetic or phenotypic effects, altered nutritional composition, environmental consequences, animal welfare issues, and uncertainties associated with long-term or large-scale deployment. Public acceptance varies substantially according to geographical region, application, cultural and ethical considerations, regulatory environment, scientific literacy, and institutional trust. Overall, GM and gene-edited organisms should not be considered a homogeneous category. Their benefits, risks, and societal acceptability depend on the specific organism, genetic modification, intended trait, and context of use, supporting a balanced, evidence-based, and case-specific approach. Full article

The emergence of seekRNA- and bridgeRNA-guided recombination has introduced a distinct paradigm in genome engineering by coupling programmable RNA-directed DNA recognition with recombinase-mediated insertion, excision, inversion, and genomic rearrangement without canonical double-strand breaks. Since their discovery in 2024, these systems have progressed rapidly from bacterial mobile genetic elements and mechanistic characterization to structural elucidation and programmable genome engineering in human cells. However, these advances remain distributed across foundational and rapidly emerging studies, creating a need for an integrated molecular perspective on their mechanisms, technological development, and position within contemporary genome engineering. This review synthesizes the molecular architecture, RNA-guided recognition, strand-exchange mechanisms, programmability, and engineering of seekRNA and bridgeRNA systems, with particular emphasis on complementary human-cell advances involving ISCro4 and engineered IS621. Whereas ISCro4 systems have enabled multikilobase DNA insertion, genomic excision, and near-megabase inversion, the enIS621–tebRNA platform has enabled scarless kilobase-scale integration across multiple human cell types, including proof-of-concept functional CD19 chimeric antigen receptor and factor IX gene insertion. The review further integrates recent genome-scale bacterial rewriting and Targetable Recombinase Assisted DNA Exchange (TRADE)-mediated DNA replacement, while benchmarking RNA-guided recombination against conventional site-specific recombination, clustered regularly interspaced short palindromic repeats (CRISPR)-based editing, Programmable Addition via Site-specific Targeting Elements (PASTE), CRISPR-associated transposases, and emerging large-payload genome-writing strategies, including kilobase-scale nickase-targeting (KNIT) editing, Prime Assembly, engineered R2 retrotransposons, and TransCRISTI. This comparative framework highlights a broader transition from programmable sequence modification toward direct engineering of genomic architecture, while identifying recognition-site constraints, mismatch-tolerant recombination, unintended recombination products, delivery, and genome-wide specificity as key translational barriers. By integrating foundational mechanisms with recent mammalian engineering, genome-scale bacterial rewriting, large-payload technologies, and emerging computational design strategies, this review provides a contemporary framework for defining the distinctive capabilities, current limitations, and future development of programmable RNA-guided DNA recombination. Full article

Malnutrition and climate-induced stress remain major constraints to global food and nutritional security despite the yield gains of the Green Revolution. Solanaceae crops such as tomato, potato, brinjal, and pepper are key sources of vitamins, minerals, and bioactive compounds. Yet, their genetic improvement has been limited by narrow diversity and complex polygenic traits. The advent of CRISPR/Cas-mediated genome editing provides a transformative platform for precision crop improvement by enabling targeted modification of genes controlling stress tolerance, yield, and nutritional quality. In Solanaceae, CRISPR/Cas applications have successfully enhanced resistance against major pathogens (SlMlo1, SlPelo, SlDCL2), improved abiotic stress tolerance through editing of SlMAPK3, SlCBF1, and SlBZR1, and optimized fruit quality traits via modulation of Psy1, CrtR-b2, and fiAD2/3. Emerging innovations, such as base and prime editing, and RNP-mediated transgene-free delivery, are expanding the precision and scope of editing. However, challenges persist, including genotype-dependent transformation, low HDR efficiency, and incomplete understanding of off-target and epigenetic effects. Integrating CRISPR with omics-guided gene discovery, efficient transformation systems, and regulatory harmonization can accelerate the development of nutritionally enriched, stress-resilient, and sustainable Solanaceae varieties. This review synthesizes recent advances, identifies critical limitations, and outlines future opportunities for deploying CRISPR/Cas technology to achieve next-generation breeding and food system resilience. Full article

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system began as a tool for programmable genome editing. CRISPR/Cas technologies have evolved into a versatile platform for functional genomic screening, epigenome editing, therapeutic target discovery, and highly sensitive molecular diagnostics. New effectors and engineered variants continue to push these applications into personalized medicine and point-of-care testing. Here, this review highlights recent advances in therapeutic target discovery and therapeutic and molecular diagnostic development utilizing CRISPR/Cas technologies. We discuss how CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), base editing, and prime editing have improved our understanding of disease mechanisms, while creating new opportunities for therapeutic intervention. Current applications in cancer immunotherapy, infectious disease, and neurological disorders are also discussed. In diagnostics, CRISPR-based platforms enable sensitive detection of infectious pathogens, cancer biomarkers, and genetic disorders using programmable nuclease activity in both laboratory and point-of-care settings. Collectively, these advances continue to expand the role of CRISPR/Cas technology across biological discovery, disease diagnostics, and therapeutic development, driving progress in precision medicine. Full article

Water deficit severely limits rice productivity. The elite Dian (D1)-type hybrid japonica rice ‘Dianheyou 615 (ZH1)’ exhibits exceptional drought adaptation in high-altitude rainfed uplands of the Yungui Plateau, yet the underlying molecular mechanisms remain unknown. We compared phenotypic and transcriptomic responses of ZH1 and six other japonica cultivars under well-watered and water-deficit conditions. Water-deficit stress significantly impaired agronomic traits across all cultivars; however, ZH1 uniquely maintained relatively stable flag leaf morphology and seed-setting rate, and displayed distinctive stomatal traits, in stark contrast to its parental lines and other cultivars. Transcriptomic profiling at the jointing-to-booting stage defined a core drought response module of 174 conserved genes across all cultivars. Critically, by intersecting 1097 ZH1-specific genes with drought-responsive elements, we pinpointed 15 core, cultivar-specific regulatory genes. These candidates are enriched in functions related to cuticle formation, carbohydrate metabolism, and stress signaling; among them, a DREB transcription factor (LOC4347618) is a prime candidate. qRT-PCR validated their expression. Using CRISPR/Cas9-mediated gene editing, we generated homozygous knockout mutants for LOC4333842, LOC4347618, and LOC4328441. Under 20% PEG-6000-simulated drought stress, all three mutant lines showed significantly increased drought susceptibility relative to wild-type controls, confirming the positive regulatory roles of these genes in drought stress tolerance in japonica rice. These results establish these three genes as promising targets for molecular breeding aimed at enhancing drought resistance in rice. Full article

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish dystrophin expression. Prime editing enables precise genome modification without generating double-strand DNA breaks or requiring donor DNA templates. We established an in vitro prime editing workflow to generate and subsequently correct the mdx5cv mutation in mouse C2C12 myoblasts. Following optimization of engineered prime editing guide RNAs (epegRNAs) and PAM-flexible prime editors, wild-type cells were edited, clonally isolated, and genotyped. Mutation correction was then evaluated using optimized epegRNA designs. Two rounds of prime editing introduced the mdx5cv mutation into approximately 20% of alleles in C2C12 cells creating the mdx5cv C2C12 cell line. Clonal isolation yielded five homozygous mutant clones among 59 expanded colonies. Optimization studies identified an epegRNA containing a 16 nucleotide reverse transcription template and a 10 nucleotide primer binding site (RTT16/PBS10) as the most efficient design. Correction of the pathogenic allele reached approximately 26%, whereas longer PBS lengths reduced editing efficiency. In silico off-target analysis using Cas-OFFinder identified no candidate genomic loci with fewer than three mismatches for the spacer sequences used in either mutation generation or correction, suggesting a favorable predicted specificity profile. Corrected mdx5cv C2C12 myoblasts retained their capacity to differentiate into multinucleated myotubes. Representative Western blot analysis detected dystrophin protein expression in differentiated corrected mdx5cv myotubes, consistent with successful correction of the pathogenic mutation. These findings establish a robust prime editing platform for both the generation and correction of the mdx5cv mutation and provide proof of concept that precise correction of the pathogenic mutation is associated with restoration of dystrophin expression following myogenic differentiation. Full article

Therapeutic cancer vaccines are designed to initiate tumor-specific immunity, yet their clinical success depends not only on antigen selection but also on the capacity to overcome the profoundly suppressive tumor microenvironment. Within tumors, abnormal vasculature, hypoxia, nutrient competition, acidic pH, and suppressive myeloid and stromal cells collectively constrain antigen presentation, T-cell priming, trafficking, and effector function, often converting otherwise immunogenic vaccination into an ineffective immune stimulus. Recent advances in neoantigen discovery, dendritic cell engineering, and nucleic acid-based vaccine platforms have improved the precision of antigen delivery, but these gains remain insufficient unless vaccine-induced responses can be sustained and executed within the hostile metabolic and immunologic landscape of the tumor niche. In this context, the tumor microenvironment is not merely a barrier to be overcome, but an active determinant of vaccine outcome that shapes immune editing, promotes exhaustion, and limits intratumoral expansion of cytotoxic lymphocytes. Accordingly, the most promising therapeutic strategies now combine vaccination with checkpoint blockade, radiotherapy, stromal remodeling, or metabolic reprogramming to recondition the tumor ecosystem and permit productive antitumor immunity. Here, we discuss how tumor microenvironmental constraints govern vaccine performance, review emerging platform technologies, and outline combinatorial strategies aimed at converting immune priming into durable tumor control. Full article

Background/Objectives: NF1 encodes neurofibromin, a RAS-GTPase-activating protein (GAP), and heterozygous loss-of-function variants cause neurofibromatosis type 1. Missense variants outside the GAP-related domain (GRD) are difficult to classify because supporting functional evidence is limited. NF1 c.6394T>C (p.Ser2132Pro) is currently listed in ClinVar as a variant of uncertain significance. We examined its functional consequences and evaluated whether the resulting evidence supports a likely pathogenic interpretation under the ACMG/AMP framework. Methods: We evaluated p.Ser2132Pro using population databases, evolutionary conservation, calibrated in silico predictors, and structural mapping onto the full-length cryo-EM model of neurofibromin. The variant was then introduced at the endogenous NF1 locus in HEK293T and A375 cells by prime editing, and we measured neurofibromin abundance, transcript levels, RAS-GTP dynamics, and MAPK pathway reactivation after PLX4032 treatment. Evidence was integrated under the ACMG/AMP framework. Results: p.Ser2132Pro was absent from population databases, affected a highly conserved residue buried within the C-terminal HEAT domain, and received concordant deleterious predictions from calibrated in silico tools. At the endogenous locus, p.Ser2132Pro reduced neurofibromin abundance by 88–95% in both cell models while NF1 transcript levels were only modestly reduced, impaired RAS-GTP signal termination, doubled steady-state RAS-GTP in A375 cells, and produced 5-fold higher ERK phosphorylation than the non-targeting control under PLX4032 treatment. Conclusions: These findings support a cellular loss-of-function effect of p.Ser2132Pro characterized primarily by reduced neurofibromin abundance with impaired neurofibromin-dependent RAS regulation. Under a conservative ACMG/AMP interpretation, the combined evidence supports a Likely Pathogenic interpretation of NF1 c.6394T>C (p.Ser2132Pro). Full article

Inherited liver diseases are predominantly caused by monogenic mutations, and the vast majority of these conditions currently lack curative treatment options. Although liver transplantation may be used for patients with end-stage disease, it faces numerous challenges, including donor organ shortage, immune rejection, and the need for lifelong immunosuppression. In recent years, CRISPR-Cas9-based gene editing technology has advanced rapidly, offering transformative hope for the treatment of these diseases. This review systematically elucidates the working principles and technical advantages of the CRISPR-Cas9 system and its derived tools (base editing and prime editing), summarizes recent applications of these technologies in the treatment of hereditary liver diseases, and discusses the prospects and challenges of their clinical translation, aiming to provide a theoretical reference for future research in this field. Full article

Root-knot nematodes (RKNs; Meloidogyne spp.) are among the most destructive plant parasites, causing severe yield losses in diverse crops. Reactive oxygen species (ROS), particularly superoxide radicals (O₂•⁻) and hydrogen peroxide (H₂O₂), are central regulators of plant–RKN interactions. This review synthesizes current molecular, biochemical, genetic, transcriptomic, and translational evidence showing that the outcome of infection is determined by the spatiotemporal regulation of H₂O₂ rather than by ROS abundance alone. In resistant interactions, nematode perception activates PTI-associated signaling through selected cell-surface receptor complexes, including some BAK1/SERK3-associated pathways, together with BIK1, Ca²⁺ signaling, and RBOHD/F, generating a sustained oxidative activity associated with salicylic acid-dependent immune signaling and reduced H₂O₂-scavenging capacity and coupled to hypersensitive response, lignin and callose deposition, and feeding site restriction. In susceptible interactions, RKNs deploy ROS-targeting effectors such as Mi-CRT, MjTTL5, CATLe, Mj-NEROSs, and CMII to suppress ROS production, enhance antioxidant scavenging, or weaken SA-dependent defense. Evidence from a cyst-nematode system suggests that RBOH-derived ROS can restrict excessive cell death around syncytia; whether an analogous lower-redox requirement exists in RKN-induced giant cells remains unresolved. Finally, redox-based strategies, including CRISPR/Cas editing, host-induced gene silencing, chemical priming, and biocontrol, are discussed as promising approaches for durable and sustainable nematode resistance. Full article

Genome editing, particularly CRISPR/Cas-based systems, has emerged as a key tool for functional genomics and trait improvement in flax (Linum usitatissimum L.), an important fiber and oilseed crop. This review focuses specifically on flax as an emerging target species and distinguishes experimentally validated applications from approaches adapted from model plants. Recent progress includes the characterization of endogenous U6 promoters, which improved guide RNA expression and contributed to enhanced genome editing performance under optimized conditions. Reported studies demonstrate efficient targeted mutagenesis in flax; however, editing outcomes remain strongly dependent on genotype, construct design, and regeneration capacity, and stable homozygous edited lines are still limited. Target genes include pathways involved in lignin and cellulose biosynthesis, fatty acid metabolism, and stress responses, influencing fiber quality, oil composition, and stress adaptation. Despite current bottlenecks such as low homologous recombination efficiency and regeneration constraints, base editing, prime editing, and multiplex CRISPR systems provide promising avenues for precision breeding in flax. Full article

Soybean is a strategic crop for global protein and vegetable oil supply chains; however, genetic improvement remains constrained by genotype-dependent regeneration, variable transformation efficiency, and regulatory concerns regarding stable transgene integration. This review synthesizes emerging DNA-free and genotype-independent genome-editing frameworks for soybean, where genotype independence is defined as the ability to recover fertile, non-chimeric edited plants across elite germplasm. We critically examine the soybean genome-editing toolbox, including CRISPR-Cas9, Cas12a, multiplex editing systems, base editing, and prime editing, and discuss persistent bottlenecks associated with target selection, off-target assessment, editability, and plant recovery. Particular emphasis is placed on artificial intelligence (AI)-assisted approaches that integrate genomic, epigenomic, chromatin-accessibility, and multi-omics datasets to improve target prioritization, guide RNA design, off-target prediction, and locus- and genotype-specific editability assessment. We further evaluate DNA-free genome-editing technologies, including CRISPR-Cas ribonucleoproteins, transient RNA-based systems, and nanocarrier-mediated delivery platforms, highlighting their potential to generate non-integrative edits while reducing prolonged nuclease exposure. In addition, we discuss regeneration reprogramming strategies based on developmental regulators and morphogenic modules, including BBM-WUS, GRF-GIF, de novo meristem induction, and somatic embryogenesis, as enabling technologies for overcoming cultivar-dependent regeneration barriers. Importantly, this review proposes an integrated AI-to-field framework that connects target discovery, editability prediction, DNA-free editing, regeneration reprogramming, phenotypic validation, and breeding deployment into a unified soybean improvement pipeline. We further highlight emerging opportunities in multi-omics-guided target discovery, genotype-aware prediction models, regeneration-aware editing strategies, and closed-loop machine-learning systems that continuously improve editing decisions through experimental feedback. Collectively, these convergent innovations provide a practical foundation for accelerating the development of climate-resilient, nutritionally enhanced, and industry-ready soybean cultivars. Full article

Nanotechnology has become an important platform in the fields of gene therapy and genome editing, providing delivery strategies that address persistent therapeutic challenges by improving the precision, efficiency, and safety of genetic modifications. This review highlights the central role of nanomaterials in overcoming persistent barriers to genetic interventions, including inefficient delivery, instability of genetic cargo, and off-target effects. Specifically, we emphasize the combined use of nanomaterials with clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems, which can improve editing specificity and therapeutic efficacy. Beyond the classical CRISPR/Cas9 platform, this review also discusses next-generation modalities such as base editors, Cas13, prime editing, and the recently described Tandem Interspaced Guide RNA and TIGR-associated protein (TIGR-Tas) system, while considering their therapeutic potential and distinct delivery challenges. By using nanomaterials, the stability and intracellular delivery of genome-editing systems are improved, enabling more effective treatments for genetic disorders and acquired diseases such as cancer and infectious diseases. In addition, nanocarriers provide controlled release, protection from degradation, and better biocompatibility, thereby improving the safety and reliability of gene-editing therapies. Despite these advances, important translational challenges remain, including immunotoxicity, large-scale manufacturing, and regulatory integration. Overall, the continued convergence of nanotechnology and genome engineering may support the development of personalized medicine strategies that adapt genetic engineering tools for patient-specific applications. Full article

Ischemic vascular diseases remain a leading cause of morbidity and mortality worldwide and are frequently associated with irreversible tissue damage. Although stem cell-based therapies have shown promise for vascular regeneration, their clinical translation has been limited by poor survival, insufficient engraftment, functional heterogeneity, and immune rejection. Recent advances in genome-editing technologies, including CRISPR/Cas9, base editing, and prime editing, have provided powerful tools for overcoming these limitations through precise genetic modification of stem cells. Gene editing can enhance angiogenic potential, improve resistance to ischemic stress, augment paracrine activity, promote endothelial maturation, and reduce immunogenicity. In this review, we outline the current genome-editing toolbox and its application to stem cell engineering for vascular regeneration in ischemic disease. We also examine emerging therapeutic concepts, universal donor cell platforms, and key issues in safety and ethics, with a focus on translational pathways. Taken together, advances at the interface of genome editing and stem cell biology are likely to accelerate the development of regenerative therapies that deliver more durable vascular repair in ischemic vascular disease. Full article