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48 records · Page 3Linked to original sources
PubMed2026

Hormone priming and metabolic engineering of phytohormone crosstalk in rice under combined biotic and abiotic stresses: a multi-omics perspective for climate-resilient crop development.

Rice (Oryza sativa L.) is the caloric backbone for more than half of humanity, yet it remains one of the most vulnerable crops to the simultaneous biotic and abiotic stresses exacerbated by climate change. Phytohormone priming and the complex crosstalk networks governed by transcription factor hubs like WRKY, MYB, and NAC serve as the central adaptive mechanism for stress resilience. This review synthesizes how multi-omics integration, including spatial and single-cell transcriptomics, is resolving the molecular architecture of hormonal priming and epigenetic stress memory. We critically evaluate advanced metabolic engineering and genome-editing strategies such as CRISPR-Cas9, base/prime editing, and synthetic gene circuits that enable precision modifications to decouple stress tolerance from historical yield penalties. Furthermore, we discuss the emerging roles of microbiome-assisted priming via synthetic consortia and the application of artificial intelligence and digital twins (continuously updated computational models of crop physiology) for predictive stress management. By integrating these diverse technological pillars, we propose a systems-level roadmap for developing climate-resilient rice cultivars capable of maintaining yield stability across a volatile combinatorial stress landscape. This synthesis provides a framework for translating mechanistic hormonal insights into field-applicable cultivars to ensure global food security.

CRISPR↗
PubMed2026

Tracking-seq: a universal off-target detection approach for CRISPR-Cas genome editing.

Tracking-seq is a highly sensitive method for genome-wide detection of off-target effects in cells edited with diverse genome editing modalities, including Cas9, cytosine base editors, adenine base editors and prime editors. Since most genome editors induce DNA repair pathways and generate single-stranded DNA (ssDNA) intermediates, Tracking-seq leverages this process by tracking replication protein A-a key protein that binds and protects ssDNA-to identify on-target and off-target events. Here we provide a detailed protocol for Tracking-seq, covering genome editing of cells, extraction of replication protein A-bound ssDNA, sequencing library construction and data analysis using our custom computational tool Offtracker. Tracking-seq is applicable to various genome editing scenarios with low cell input, delivering high-performance results. The entire workflow, from genome editing to data analysis, can be completed within 1-2 weeks, making it a rapid solution for assessing genome-wide off-target activity.

CRISPR-Cas Systems↗
PubMed2026

A voyage of reprogrammable metabolic bioengineering reshapes plant defense: from editing tools to synthetic systems.

Metabolic bioengineering has emerged as a transformative approach for reshaping plant defense by targeting intrinsic biosynthetic pathways to enhance immunity in modern agriculture. Moving beyond proof-of-concept metabolomics to broad-spectrum programmable pathway engineering addresses gaps in plant rational design and optimizes resilience in response to diverse environmental cues. This review aims to comprehensively highlight the transition of innovative approaches to phenolics, alkaloids, flavonoids, terpenoids, and benzoxazinoids, inferring adaptive reprogramming that mediates the growth-defense balance and functions as molecular sentinels in plants. Furthermore, decoding the volatile metabolome reveals a dynamic signaling interface that influences defense responses and stress-induced plant-microbe interactions, with the shikimate, jasmonate, and salicylate pathways functioning as central hubs for microbial deterrence and priming immune memory. Recent developments in multi-scalar genome-editing strategies, including CRISPR-driven combinatorial edits, enzyme orthogonalization, fluxomics, and spatially resolved multi-omics, reconfigure central and specialized metabolic fluxes toward improved defense function and regulation. Additionally, emerging tools, such as WUSCHEL2 and BABY BOOM transcriptional modules, and artificial engineering strategies integrating deep learning model-driven predictions facilitate rapid development of synthetic genetic circuits and support a predictive engineering of plants. Moreover, Mass spectrometry imaging (MSI) in spatial metabolomics enables to obtain structures and locations of unidentified endogenous metabolites within cells and tissues. Overall, this review emphasizes a diverse array of primary and secondary metabolites, spanning molecular concepts to recent advances in plant immune mechanisms. It also illustrates new frontiers in programmable metabolic engineering that accelerate the understanding of plant-microbe-metabolite cross-talks, offering strategies to improve plant resistance and advance sustainable agricultural solutions.

metabolic bioengineering↗
PubMed2026

Epigenetic priming and locus-specific demethylation enhance cell-death susceptibility in liver cancer.

Liver cancer treatment with epigenetic drugs remains challenging because demethylating agents such as 5-azacytidine (5-AZA) induce genome-wide toxicity and may activate oncogenes. We hypothesized that a low, nontoxic dose of 5-AZA could prime liver cancer cells by partially relaxing chromatin at selected loci to restore silenced cell-death regulators. HepG2 cells treated with 2 μM 5-AZA underwent ATAC-seq and RNA-seq to identify genes with promoter opening and increased expression. Among ten candidates, BFL-1 and SQOR were prioritized for roles in cell death and redox control. Forced expression of either gene increased sensitivity to TNF-α/cycloheximide (CHX) and sorafenib, both of which elevated mitochondrial reactive oxygen species. To establish causality in a physiological context, we used CRISPR-dCas9-TET1 to demethylate CpG-rich promoter regions of BFL-1 or SQOR. Locus-specific editing sensitized cells to TNF-α/CHX more rapidly than conventional overexpression and reproduced the heightened death response elicited by low-dose 5-AZA without baseline toxicity. Analysis of the cancer cell line encyclopedia and The Cancer Genome Atlas datasets showed consistent BFL-1 downregulation in liver cancer, variable SQOR expression across cancers, and positive correlations of both genes with tumor-suppression markers and immune-cell infiltration. These results indicate that targeted reactivation of BFL-1 and SQOR increases cell-death susceptibility in liver cancer cells. Integrating low-dose pharmacologic priming with precise epigenetic editing may preserve genome-wide methylation while restoring cell-death competence, providing proof-of-concept for locus-specific epigenetic therapy in liver cancer.

Humans↗
PubMed2026

Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of capping protein and VASP.

Cell migration frequently employs protrusions termed lamellipodia, constituting the prime model system for generation of branched actin filament networks. Here we utilize genome editing to explore the functional connections between the actin monomer-binding protein profilin (Pfn), the filament nucleating Arp2/3 complex, its co-factor heterodimeric capping protein (CP) and Ena/VASP family polymerases in lamellipodial actin assembly. We show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. While Pfn promotes Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal vanishes in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, differentially regulating VASP and its antagonizer CP. Mathematical modeling of our data suggest Ena/VASP and CP to compete for recruitment to lamellipodial edges. Our work provides critical insights into the molecular logic of branched actin network assembly in protrusion and force generation.

Profilins↗
PubMed2025

Refined and benchmarked homemade media for cost-effective, weekend-free human pluripotent stem cell culture.

BACKGROUND: Cost-effective, practical, and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However, the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover, we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly, the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation. METHODS: We formulated B8 with commercially available, growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made, weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry, and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability, genome editing efficiency, single-cell cloning, and differentiation in both monolayer and organoids. Finally, we validated key findings using male (H1) and female (H9) human embryonic stem cells. RESULTS: hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast, cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time, B8+ led to gene expression changes indicative of marked lineage priming, reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state, consistent with intermittent media starvation. CONCLUSIONS: Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8, integrating some features of B8 while resembling cE8, emerges as a robust and practical option with limited compromises. B8+, though advantageous in some contexts, warrants caution due to lineage priming effects that may impact differentiation outcomes.

hiPSC; pluripotency; culture media; thermostable F↗
PubMed2026

Precision Engineering of Evolution-Resilient Rice against Bacterial Blight.

The persistent conflict between rice and Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight, exemplifies a dynamic genetic arms race in agriculture. The cyclical deployment and erosion of major resistance (R) genes highlight the high adaptive potential of Xoo and the need for strategies that are durable rather than absolute. This review synthesizes a paradigm shift from reactive, single R-gene deployment toward proactive engineering of evolution-resilient resistance. We explore the molecular-genetic basis of Xoo adaptability, including TAL effector diversification, non-TAL virulence functions, genome variation, and immune suppression mechanisms. In response, we propose a framework for durable disease management with three connected components: precision disarmament through editing of susceptibility-gene effector-binding elements and executor/decoy designs; smart induction through targeted delivery and immune priming; and ecological fortification through protective microbiomes. We also discuss the limits, trade-offs, and field-validation requirements of these approaches. Integrating frontier technologies with evolutionary genetics, predictive genomics, and pathogen population dynamics can help develop rice varieties and deployment systems that are more difficult for Xoo populations to overcome.

CRISPR↗
PubMed2025

Cell-type-specific response to silicon treatment in soybean leaves revealed by single-nucleus RNA sequencing and targeted gene editing.

Mineral nutrient uptake and deposition profoundly influence plant development, stress resilience, and productivity. Silicon (Si), though classified as a non-essential element, significantly influences a plant's physiology, particularly in fortifying defense responses and mitigating stress. While the genetic and molecular mechanisms of Si uptake and transport are well studied in monocots, particularly rice, their role in dicot species, such as soybean, remains unclear at the cellular and molecular levels. In this study, we utilized single-nucleus RNA sequencing (snRNA-seq) to dissect cellular responses to Si accumulation in soybean leaves. We identified distinct cellular populations, including a unique Si-induced or Si-associated cell cluster within vascular cells, suggesting a specialized mechanism of Si distribution. Si treatment notably induced the expression of defense-related genes, with a pronounced enrichment in vascular cells, underscoring their pivotal role in activating plant defense mechanisms. Moreover, Si modulated the expression of genes involved in phytoalexin biosynthesis, salicylic acid, and immune receptor signaling, suggesting transcriptional priming of genes involved in defense responses. Further investigation of Si transporters revealed precise expression of an Si efflux gene in epidermal cells in response to Si treatment. We also validated the role of efflux Si transporters using a Xenopus oocyte assay and CRISPR/Cas9 genome editing of composite soybean plant roots. This study provides critical insights into the biotic stress regulatory networks influenced by Si treatment in soybean leaves at the single-cell level, thus laying the foundation for enhancing stress tolerance through optimized mineral nutrient uptake.

Glycine max↗
PubMed2026

Gene therapy for genodermatoses at the crossroads of innovation and clinical translation.

Inherited genodermatoses are a heterogeneous group of rare monogenic disorders. Among these, epidermolysis bullosa (EB) and ichthyoses represent paradigmatic disorders characterized by severe skin fragility and hyperkeratosis, respectively, and impaired barrier function, often with profound effects on quality of life and systemic health. Current management remains largely palliative, underscoring the urgent need for disease-modifying therapies. Over the past 2 decades, advances in epithelial stem cell biology, vector engineering and genome editing technologies have transformed the therapeutic landscape for genodermatoses. Ex vivo gene therapy has provided the first proof that genetically corrected epidermal stem cells can achieve long-term tissue regeneration in EB skin patients, establishing a new paradigm for regenerative medicine. In parallel, the emergence of programmable genome engineering platforms, including CRISPR/Cas nucleases, base editors and prime editors, have enabled increasingly precise strategies for mutation-specific correction in both recessive and dominant disorders. Furthermore, the development of in vivo topical approaches is expanding the possibility of directly targeting the skin. Despite these advances, substantial translational barriers continue to limit broad clinical implementation. Efficient and durable targeting of epidermal stem cells within a highly regenerative tissue, together with safe delivery across the skin barrier, stringent control of off-target activity, scalable manufacturing and demonstration of long-term safety, remain major challenges for the clinical translation of these approaches. In this Review, we discuss the current state of gene therapy for genodermatoses, highlighting key clinical milestones, emerging genome editing technologies and next-generation delivery systems. We further examine the biological and regulatory challenges that need to be overcome to bridge the gap between experimental innovation and clinically accessible therapies for patients with inherited skin diseases.

epidermolysis bullosa (EB)↗
PubMed2026

Humanizing acidic mammalian chitinase variants establish lung immune conditioning and control environmentally driven inflammation and fibrosis.

Chitin, a widespread environmental particle constituent, triggers lung inflammation but is degraded by chitinases. In humans, single-nucleotide polymorphisms (SNPs) in CHIA (acidic mammalian chitinase; AMCase) are associated with lung disease, suggesting that chitinase variants influence responses to airborne particles. Here, we edit the mouse Chia1 locus to generate humanized (hChia) mice harboring common human SNPs. Compared with controls expressing disease-protective SNPs, hChia mice lack robust chitinase activity and fail to degrade natural chitin substrates. Lung-resident lymphocytes and macrophages are spontaneously primed and sensitive to inflammatory triggering by environmental chitin. Immune cell infiltration correlates with airway chitin following challenge, and hChia mice exhibit exacerbated inflammatory and fibrotic lung disease. In humans with acute respiratory failure, alveolar hemorrhage coincides with environmentally derived chitin particles that are susceptible to chitinase degradation, attenuating inflammatory cell responses. Thus, environmental chitin and chitinase activity are crucial determinants of lung immune conditioning with potential therapeutic applications.

AMCase↗
PubMed2026

Ultra-sensitive profiling of CRISPR-Cas off-target effects with Tracking-seq2.

Accurate detection of off-target activity in primary human cells is crucial for ensuring the safety of gene therapies, yet existing methods often lack sufficient sensitivity. To address this limitation, we develop Tracking-seq2, an advanced technology that integrates exogenous 5' → 3' exonuclease treatment and non-homologous end joining (NHEJ) pathway inhibitors with the original Tracking-seq. Tracking-seq2 exhibits enhanced sensitivity in profiling off-target sites of diverse genome editors-including Cas9, Cas12a, cytosine base editors (CBEs), adenine base editors (ABEs), and prime editors (PEs). Critically, Tracking-seq2 is directly applicable to clinically relevant primary human cell types, such as T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Furthermore, our findings reveal that genomic variations drive distinct off-target heterogeneity across different individuals, highlighting the necessity for personalized safety assessment in clinical genome editing applications. Tracking-seq2 provides a robust platform for sensitive off-target detection in primary cells, with sensitivity comparable to or exceeding current state-of-the-art methods.

Humans↗
PubMed2026

Duchenne muscular dystrophy: from gene to gene-ius therapies.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional dystrophin, leading to progressive muscle degeneration, loss of ambulation, respiratory failure, cardiomyopathy, and premature mortality. Despite advances in multidisciplinary supportive care, DMD remains an incurable disease associated with substantial physical, psychosocial, and economic burdens. The monogenic nature of DMD and its well-defined molecular pathogenesis have made it a prime target for the development of precision therapies aimed at restoring dystrophin expression or modifying disease progression. This review provides an overview of the genetic and molecular mechanisms underlying DMD, summarizes its clinical manifestations and natural history, and discusses current standards of care. It further examines recent advances in disease-modifying therapeutic strategies, including exon-skipping antisense oligonucleotides, nonsense mutation readthrough agents, adeno-associated virus (AAV)-mediated micro-dystrophin gene replacement, and emerging genome-editing technologies such as CRISPR/Cas9. The review also highlights the limitations of existing treatments, including mutation specificity, variable efficacy, immune-related challenges, and uncertainties regarding long-term durability and safety. Finally, it considers future directions in therapeutic development, emphasizing the need for combination approaches, improved delivery systems, and next-generation gene-editing platforms to achieve more effective and lasting clinical outcomes. Collectively, these advances represent a paradigm shift in DMD management and offer renewed hope for improving survival and quality of life for affected individuals.

Humans↗