Abstract Background Fulfilling the promise of human genetics in elucidating disease requires identifying causal variants and genes underlying genetic association signals. Molecular quantitative trait locus (molQTL) analyses, e.g. expression QTL (eQTL) and splicing QTL (sQTL), link genetic variants to intermediate molecular phenotypes, but pinpointing causal variants and their regulatory effects remains challenging. Here, we integrate sQTL analysis with deep-learning-based splicing effect annotation to identify causal genetic variants and elucidate their functional mechanisms affecting human phenotypes. Results Using a single-cell GWAS method (scHi-HOST) on 96 lymphoblastoid cell lines (LCLs) with and without influenza A virus (IAV) infection, we discovered ~ 43,000 sQTLs associated with 217 genes after IAV infection. Integrating sQTLs with AI splice prediction, we uncovered 76 likely causal variants that affect cis-acting molecular splicing components (5’ donor, 3’ acceptor), supported by further computational analysis. Among these, we experimentally validated a causal sQTL signal affecting poly (ADP-ribose) polymerase 2 (PARP2). The causal variant, rs2297616, alters the 5’ splice donor site in the second intron of PARP2 , resulting in two protein isoforms differing by 13 amino acids. The derived A allele was associated with the longer protein isoform and increased IAV levels in LCLs. CRISPR editing validated the causal effect of this variant on both protein length and IAV infection. Lastly, these 76 putative causal sQTLs were further linked to over a hundred GWAS traits, including many variants associated with autoimmune diseases. Conclusions Our work provides a catalog of causal sQTL with direct splicing impacts, providing causal mechanistic insights from genotype to disease susceptibility.

Spinal cord regeneration requires a transformative strategy capable of rewriting inhibitory genetic programs while orchestrating real-time electrical communication with regenerating neural tissues. Recent advancements in precision CRISPR genome editing effectively silence or activate crucial molecular gatekeepers such as PTEN, SOCS3, and various epigenetic repressors, thereby reactivating dormant intrinsic regenerative pathways and enabling robust axonal growth. Concurrently, cutting-edge bioelectronic technologies utilizing piezoelectric, triboelectric, and magnetoelectric scaffolds have emerged, adeptly harnessing the body's inherent biomechanical energy. These innovative materials convert subtle physiological micromotions into finely tuned electrical stimuli, precisely guiding neuronal regeneration without external power sources, addressing limitations associated with traditional implants such as infection risks and mechanical incompatibility.Integrating these genetic modifications with bioelectric innovations creates a potent synergy. Genome-level reprogramming amplifies neuronal responsiveness to bioelectrical signals, markedly enhancing axonal regeneration. Simultaneously, autonomous electrical stimulation sustains and stabilizes cellular, metabolic, and synaptic improvements induced by genomic interventions, forming a closed-loop, self-sustaining therapeutic platform. This advanced system significantly transcends conventional transient recovery approaches, moving toward durable, personalized outcomes. Such convergence of advanced genetic engineering and intelligent biomaterial design represents a groundbreaking shift in regenerative neurology.Despite promising preclinical outcomes, significant translational challenges remain. Critical hurdles include ensuring precise delivery of CRISPR tools, mitigating off-target genomic effects, enhancing biocompatibility and scaffold stability, and navigating rigorous regulatory pathways. Addressing these challenges necessitates integrating next-generation gene-editing technologies, comprehensive genomic surveillance, advanced biomaterial sciences, and meticulous preclinical evaluations. Future directions in spinal cord injury research encompass multiplex genome editing, AI-driven scaffold optimization via digital twins, and tailored immune-evasive biomaterials. Collectively, this innovative approach has the potential to redefine regenerative medicine's boundaries, offering unprecedented hope for sustained, personalized recovery and dramatically improving quality of life for individuals affected by spinal cord injuries.

Abstract Background Heterozygous variants in CTNND2 , encoding the brain-specific protein δ-catenin, are associated with a broad spectrum of neurodevelopmental disorders, including dyslexia, attention deficit hyperactivity disorder, intellectual disability, and autism. Despite its clinical significance, the full phenotypic spectrum of CTNND2 -associated disorders and the neurodevelopmental role of δ-catenin, a key component of the cadherin-catenin cell adhesion complex, remain poorly defined. Methods Through international collaboration, we assembled the phenotypic and molecular information for 57 individuals, 42 previously unpublished, carrying heterozygous CTNND2 variants. All individuals were evaluated by local clinicians, and the variants were identified through exome or genome sequencing, clinical microarray, or karyotyping. To investigate the effects of δ-catenin loss on early neurogenesis, we performed neural differentiation and transcriptomic profiling in three patient-derived neural stem cell lines and three CRISPR-Cas9-generated CTNND2 knockout lines. In one patient-derived line, we further analyzed cerebral organoid development and performed pathway modulation to assess phenotypic rescue. Results The 41 CTNND2 variants included 12 previously reported loss-of-function- and one missense variant, and 28 novel variants comprising 10 missense and 18 predicted loss-of-function changes. Eight of the novel variants occurred de novo , and 12 were inherited from a parent with a neurodevelopmental phenotype. The most common clinical features were developmental delay (90%), intellectual disability (74%), and behavioral abnormalities (79%). Functional studies revealed impaired early neurogenesis in one patient-derived line, characterized by aberrant neural rosette formation. Transcriptome analysis showed dysregulated WNT signaling, and partial rescue of these defects was achieved by modulating the WNT pathway, highlighting δ-catenin's role in early neural development. Conclusions This study defines the clinical symptoms of CTNND2 -related neurodevelopmental disorders, outlining a recognizable yet variable phenotype that overlaps with other forms of intellectual disability and autism. Our findings provide preliminary evidence of genotype–phenotype correlations and highlight δ-catenin's critical role in modulating WNT signaling during early neural development. These insights advance our understanding of CTNND2 -associated disorders and support the importance of mechanistic studies to inform personalized diagnostics and therapies.

Background: Aging brains are shaped by a persistent dialogue between declining neurogenesis and rising neuroinflammation. Neural stem cells progressively lose regenerative capacity, while microglia and astrocytes shift toward maladaptive states that erode synaptic plasticity and cognition. This convergence defines inflammaging, a slow yet relentless process that undermines resilience. However, the field remains hampered by critical gaps: incomplete mapping of microglial heterogeneity, poorly understood epigenetic scars from inflammasome signaling, lack of longitudinal data, unclear niche-specific immune mechanisms, and uncertain cross-species relevance. This review addresses these pressing barriers, aiming to transform fragmented insights into actionable strategies. Summary: I chart how neurogenesis and neuroinflammation operate in continuous dialogue, identify five major knowledge gaps, and evaluate strategies to reprogram this interaction. Approaches include longitudinal imaging, niche-focused immunomodulation, glial subtype reprogramming, brain-penetrant inflammasome inhibitors, and CRISPR-based epigenetic editing. Each strategy is mapped against translational potential, short-term feasibility, and long-term vision, with emphasis on how mechanistic precision can guide clinical innovation. Conclusion: Here I highlight that neurogenic potential is not entirely lost with age but may be preserved or restored by tuning immune and epigenetic environments. This review proposes a roadmap for reshaping the aging brain’s fate, offering mechanistically grounded strategies to delay cognitive decline. Beyond neurology, the work underscores a broader principle: by integrating cellular plasticity with immune modulation, science edges closer to re-engineering resilience across the lifespan.

The Hedgehog (Hh) signaling pathway is a key regulator of adipogenesis and lipid metabolism. However, the specific role of its receptor, Patched2 (Ptch2), in these processes remains unknown. Here, using a CRISPR/Cas9-mediated ptch2 homozygous mutation model in Nile tilapia (Oreochromis niloticus), we found that Ptch2 deficiency induced visceral and perirenal lipomatosis characterized by small, multinucleated adipocytes. Comparative adipose transcriptomics revealed pronounced adipogenic reprogramming, with marked upregulation of genes governing de novo lipogenesis (e.g., acaca, fasn), fatty acid desaturation (e.g., scd, fadsd6), and triglyceride synthesis (e.g., dgat2, lpl). Biochemically, mutants exhibited elevated blood glucose and liver transaminases (alanine aminotransferase, aspartate aminotransferase) with reduced alkaline phosphatase, indicating systemic metabolic dysregulation and hepatic stress. Our findings demonstrate that loss of Ptch2 triggers lipoma formation and adipogenic transcriptome reprogramming, highlighting its essential role in maintaining adipose tissue homeostasis.

A/T(U) and G/C nucleobase pair formation in DNA and RNA are crucial to numerous fundamental biological processes, including replication, transcription, and translation. The specificity of A/T(U) and G/C base pairing is used to recognize complementary sequences in medical and biotechnological applications, such as PCR, nucleic acid drugs, and CRISPR–Cas9-based gene editing. To understand the fidelity of biological reactions and improve the accuracy and efficacy of applications, particularly by avoiding off-target binding, clarifying the mechanism of recognition of complementary bases or sequences is essential. Despite the prevailing view that Watson-Crick hydrogen bonding is a primary mechanism for complementary base recognition, several experiments have shown that DNA polymerase does not require hydrogen bonding to select complementary bases. Other factors—such as the geometry of bases and base stacking—appear to be involved in the selection. Artificial base pairs lacking hydrogen bonds but recognized by DNA polymerase were successfully designed solely based on base-pair geometry. However, hydrogen bonding also contributes to recognition. Furthermore, the accuracy of selecting a complementary nucleobase or sequence varies across reactions, suggesting the existence of multiple selection mechanisms. This review provides an overview of biological processes and applications involving base pairing and discusses the molecular mechanism underlying complementary base recognition.

Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterisation of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differ significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterisation based on ancestral spacer (AS) and TR sequences indicate that E. faecalis with the same CRISPR1-AS genotype do not always harbour same CRISPR2-AS genotypes, and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to multilocus sequence typing (MLST), suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in the Southeast Asia region.

Abstract Background Resistance to carbapenems and third-generation cephalosporins is increasing in Klebsiella pneumoniae globally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely used to circumvent β-lactamase-mediated resistance. In 2021, an unusual K. pneumoniae clinical isolate, KpMVR1, was recovered from a hospitalised patient in England, exhibiting resistance to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. To investigate this phenomenon, we characterised the genome and antimicrobial susceptibility of KpMVR1 alongside two clonally related isolates susceptible to all three β-lactam/β-lactamase inhibitor combinations: KpMVS1, collected from the same patient 42 days earlier, and KpMVS2, from another patient in the same hospital. Methods Illumina and MinION whole-genome sequencing were conducted for these three isolates, followed by hybrid genome assembly. Annotated genome assemblies were compared to identify genetic variation. Mutagenesis experiments were performed to verify predicted functional alterations. Results All isolates belonged to clone ST8134 and carried bla KPC−2 alleles (KpMVR1: bla KPC−157 ; KpMVS1 and KpMVS2: bla KPC−2 ) in presumptively conjugative plasmids. Insertion sequence IS Ec68 caused a frameshift mutation in KpMVR1’s ompK36 gene, reducing susceptibility to meropenem-vaborbactam and imipenem-relebactam. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. Conclusions KpMVR1 carried multiple resistance-associated genetic alterations and likely developed its resistance profile through within-patient evolution. This study highlights the importance of routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy and the need to characterise underlying resistance mechanisms to assess the risk of onward dissemination.

Abstract Background Cold stress severely limits the productivity and geographical distribution of mango, a tropical fruit crop of significant economic importance. We combined transcriptomic profiling and functional genomics to investigate the molecular mechanisms underlying cold tolerance in mango. Results Comparative analysis of the cold-tolerant Jinhuang (JH) and cold-sensitive Guiqi (GQ) cultivars identified 7,757 differentially expressed genes, with significant enrichment in plant hormone signal transduction pathways. Notably, the DELLA protein GAIP-B (LOC123214451) was markedly upregulated in JH under cold stress. Functional validation through CRISPR/Cas9-mediated knockout and overexpression in Nicotiana benthamiana demonstrated that GAIP-B is both necessary and sufficient for cold tolerance. The knockout lines exhibit extreme cold sensitivity, reduced antioxidant enzyme activity, increased oxidative damage, and impaired osmotic adjustment. Conversely, the overexpression lines showed enhanced cold tolerance, with superior antioxidant capacity, maintenance of photosynthetic pigments, and increased soluble sugar accumulation. Conclusions These findings establish GAIP-B as a central regulator that integrates antioxidant defense, photosynthetic protection, and osmotic adjustment in the cold stress response. This study provides valuable insight into molecular breeding strategies aimed at enhancing cold tolerance in mango and other economically important fruit crops.

Abstract K. pneumoniae strains that combine multidrug resistance, hypervirulence and persistence are spreading worldwide, causing a severe threat to public health. Unraveling the genetics that underlies these high-risk clones is critical for the development of countermeasures. We isolated K. pneumoniae JU-BAEC-01 from treated effluent of antibiotic-manufacturing pharmaceutical facilities in Bangladesh. Herein, we report a comprehensive genomic analysis of the K. pneumoniae strain JU-BAEC-01 using whole-genome sequencing, comparative genomics, and various bioinformatics tools, including CARD, ResFinder, VFDB, PADLOC, Defense Finder, and CRISPRCas Finder, to outline its phylogenetic position, antibiotic resistance profile, virulence potential, mobile genetic elements, and antiviral defense systems. JU-BAEC-01 belongs to a phylogenetically distinct lineage, serotype O3b:KL150, unrelated to globally dominant high-risk clones. This isolate shows resistance to nearly all clinically relevant antibiotic classes except carbapenems and colistin, mediated by an extensive acquired resistome, including tmexCD3-toprJ3 (tigecycline), armA, aac(6')-Ib-cr, qnrB4, oqxAB, blaDHA-1, blaSHV-182, and blaTEM-1B, mostly carried on conjugative IncC, IncFIB, IncHI1B, and IncR plasmids. Classical hypervirulence markers are present: complete aerobactin (iucABCD-iutA) and salmochelin (iroBCDEN) clusters, rmpA2, type 1 and type 3 fimbriae, T6SS, and pgaABCD. Six prophage regions and multiple insertion elements further enhance genomic plasticity. Notably, the strain encodes one of the most elaborate anti-phage defense arsenals reported in Klebsiella to date, comprising functional Type I-E, III-A, and IV-A CRISPR-Cas systems, multiple restriction-modification systems, BREX Type I, abortive infection systems (AbiE, AbiU), and additional novel defenses that coexist with phage-derived anti-CRISPR (AcrIE9) and anti-restriction (ArdA) proteins. Klebsiella pneumoniae JU-BAEC-01 is a "perfect storm" pathogen that combines pan-drug resistance (PDR), hypervirulence, and a multilayered, highly developed defense against bacteriophages. This genomic convergence confounds treatment options and emphasizes the evolutionary capability of this priority pathogen to resist both the antimicrobial and natural predatory pressures. The presence of phage anti-defense systems underlines a dynamic co-evolutionary arms race with significant implications for the potential failure of phage therapy against such robustly defended isolates.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) and next-generation al-logeneic T cell therapies are curative options for hematologic malignancies, but their effi-cacy is often limited by graft-versus-host disease (GvHD), a complex syndrome resulting from dysregulated donor–host immune interactions. GvHD is mediated by interconnected signaling pathways that regulate T cell activation, metabolic and epigenetic program-ming, tissue-specific migration, stromal-immune interactions, and fibrotic remodeling. Therapeutic agents targeting key pathways, including ruxolitinib (JAK1/2), ibrutinib (BTK/ITK), and belumosudil (ROCK2), can modulate inflammatory responses while pre-serving graft-versus-tumor (GvT) activity. Emerging technologies, such as CRISPR-based genome editing, single-cell and spatial multi-omics, and AI-driven network modeling, enable patient-specific mapping of signaling hierarchies, prediction of disease trajectories, and identification of actionable targets. Integration of these approaches supports precise modulation of immune circuits, offering alternatives to broad immunosuppression. These insights provide a framework for next-generation, individualized interventions that pro-mote durable immune tolerance without compromising anti-tumor immunity and high-light rational combination strategies to improve outcomes in allo-HSCT and allogeneic T cell therapies.

Background: /Objectives: Bacterial biofilms formed by Escherichia coli pose a significant challenge in veterinary medicine due to their intrinsic resistance to antibiotics. Antimicrobial peptides (AMPs) represent a promising alternative. AMPs exert their bactericidal activity by binding to negatively charged phospholipids in bacterial membranes via electrostatic interactions, leading to membrane disruption and rapid cell lysis. Methods: In vitro assays included MIC determination, biofilm eradication testing (crystal violet, colony counts, CLSM), swimming motility, and EPS quantification. CRISPR/Cas9 was used to construct and complement a kduD mutant. A transposon mutagenesis library was screened for biofilm-defective mutants. In vivo, a murine excisional wound infection model was treated with CRAMP-34, with wound closure and bacterial burden monitored. Gene expression changes were analyzed via RT-qPCR. Results: The mouse-derived AMP (abbreviation CRAMP-34) effectively eradicates pre-formed biofilms of a clinically relevant, porcine-origin E.coli strain and promotes wound healing in a murine infection model. We conducted a genome-wide transposon mutagenesis screen, which identified kduD, as a critical gene for robust biofilm formation. Functional characterization revealed that kduD deletion drastically impairs flagellar motility and alters exopolysaccharide production, leading to defective biofilm architecture without affecting growth. Notably, the anti-biofilm activity of CRAMP-34 phenocopied aspects of the kduD deletion, including motility inhibition and transcriptional repression of a common set of biofilm-related genes. Conclusions: The research highlight CRAMP-34 as a potent anti-biofilm agent and unveil kduD as a previously unrecognized regulator of E.coli biofilms development, whose associated pathway is implicated in the mechanism of action of CRAMP-34.

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal haematopoietic stem cell disease, characterized primarily by intravascular hemolysis, thrombosis, and bone marrow failure. Complement inhibitors are commonly used in clinical treatment, showing limited efficacy, thus highlighting the urgent need to identify new therapeutic targets and explore alternative treatment strategies to provide theoretical guidance for clinical practice. Methods: We established a PNH cell model and constructed a miRNA–mRNA regulatory network to identify key miRNAs and core target genes. Single-cell sequencing data were analyzed to further clarify the critical genes. Drug prediction was then performed using multiple databases to identify potential therapeutic agents for PNH, which were subsequently validated through molecular docking and molecular dynamics simulations. Results: Using CRISPR/RNP technology, we successfully constructed a PIGA-knockout (PIGA-KO) THP-1 cell model. Differential expression analysis identified 1979 differentially expressed mRNAs (DEmRNAs) and 97 differentially expressed miRNAs (DEmiRNAs). The multiMiR package in R was used to predict the target genes of DEmiRNAs, from which those experimentally validated through dual-luciferase reporter assays were selected. After integrating with the DEmRNAs, a miRNA–mRNA regulatory network was constructed, comprising 26 miRNAs and 38 mRNAs. Prediction of miRNA pathway enrichment analysis identified hsa-miR-23a-3p as a key miRNA, with CXCL12, CXCL8, HES1, and TRAF5 as core target genes. Integration of single-cell sequencing datasets (PRJNA1061334 and GSE157344) was performed, followed by cell communication and enrichment analysis. This approach, combined with clinical relevance, identified neutrophil cluster as a key cluster. Intersection analysis of neutrophil cluster differential analysis result with key modules from hdWGCNA further clarified the critical genes. Drug prediction using the EpiMed, CMap, and DGIdb identified Leflunomide, Dipyridamole, and Pentoxifylline as potential therapeutic agents. Molecular docking and molecular dynamics simulations showed stable binding of these potential drugs to the critical molecules, indicating a strong molecular interaction foundation. Conclusion: Leflunomide, Dipyridamole, and Pentoxifylline may serve as promising therapeutic agents for PNH, and the hsa-miR-23a-3p/CXCL8 regulatory axis could play a pivotal role in the pathogenesis and progression of PNH.

Soybean is usually grown at large scales, with pest control based on insecticides. However, the overuse of chemicals has led to several adverse effects. Thus, integrated pest management (IPM) is the best way to protect yield through integrating different pest control tools, based on plant resistance (including Bt cultivars), adoption of economic thresholds (ETs), scouting procedures, use of selective insecticides, biological control, and other sustainable tools, which help maintain environmental quality in an ecological and economical manner. Soon, those tools will also include RNAi, CRISPR based control strategies, among other sustainable alternatives. In Brazil, results from the Soybean-IPM Program indicate that adopters of the technology have reduced insecticide use by approximately 50% relative to non-adopters, with yields comparable to or slightly higher than those of non-adopters. This reduction can be explained not only by the widespread adoption of Bt soybean varieties across the country but also by the adoption of ETs in Soybean-IPM, which has reduced insecticide use, thereby increasing natural biological control in the agroecosystem. However, low refuge compliance has led to the first cases of pest resistance to Cry1Ac, thereby growing reliance on chemical control and posing an additional challenge for integrated pest management practitioners. The obstacles to adopting IPM programs for commodity crops, such as soybean, may be mitigated by recent economic incentives within the new global agenda for decarbonized agriculture and the increase of bioinputs available in the Brazilian market. Such incentives can support the broader adoption of IPM, thereby reducing dependence on chemical inputs to achieve high yields.

Programmable organoids are emerging as a powerful new class of engineered developmental systems in which genetic circuits, epigenetic memory architectures, synthetic organizers, and closed-loop control frameworks converge to enable precise regulation of morphogenesis. Traditional organoids rely on spontaneous self-organization, but this intrinsic variability limits reproducibility, causal inference, and translational relevance. Recent advances in CRISPR-based transcriptional and epigenetic engineering, optogenetic and chemogenetic patterning technologies, reaction–diffusion design, and real-time biosensing now allow developmental trajectories to be scripted with increasing precision. This review synthesizes these developments into a unified framework spanning genetic circuit construction, epigenetic programming, synthetic morphogenesis, multi-scale sensing, adaptive regulation, and AI-guided design. Applications across human developmental biology, disease modeling, and regenerative medicine are highlighted, alongside the technical, biosafety, and ethical considerations associated with building increasingly autonomous, self-regulating developmental systems. Collectively, these advances establish programmable organoids as a foundation for developmental synthetic biology and outline a roadmap toward fully engineered human developmental architectures.

Abstract WNT5A-driven Wnt/Planar Cell Polarity (Wnt/PCP) signaling is essential for vertebrate limb morphogenesis, and pathogenic WNT5A variants cause autosomal dominant Robinow Syndrome (RS). The recurrent Cys83Ser (C83S) substitution is among the best-studied RS-associated alleles and has been variably described as hypomorphic, loss-of-function, or dominant-negative based largely on overexpression in non-mammalian systems. However, a physiologically relevant mammalian model and robust in vivo readouts that distinguish loss- from gain-of-function Wnt/PCP perturbations have been lacking. Here, we introduce a quantitative image-based metric, the local misalignment score (LMS), which visualizes and measures cell orientation in situ across long bones during late embryonic development. LMS captures local coherence of chondrocyte alignment independently of embryo age, sectioning depth, or canonical Wnt activity, and selectively recognizes Wnt/PCP defects in Wnt5a and Wntless conditional knockouts, but not in Lrp5/6-deficient limbs. Using LMS, we show that a CRISPR/Cas9-engineered Wnt5a-C83S mouse exhibits profound and uniform chondrocyte orientation defects and limb shortening that are mechanistically distinct from the spatially patterned phenotypes in Wnt5a loss-of-functon(Wnt5a-cKO) and ectopic-expression (Wnt5a-LSL) models, and occur without SOX9 downregulation or severe distal digit defects. Complementary in vitro analyses using a KIF26B-NanoLuc Wnt/PCP reporter and biochemical readouts demonstrate that WNT5A-C83S has markedly reduced signaling activity but does not inhibit wild-type WNT5A in autocrine, paracrine, or juxtracrine contexts, arguing against a dominant-negative mechanism. Together, these data support a model in which WNT5A-C83S behaves as a hypomorphic, non–dominant-negative variant that perturbs Wnt/PCP gradient-dependent limb development through altered, rather than simply reduced, signaling output. More broadly, LMS provides a scalable morphological readout for spatially resolved interrogation of Wnt/PCP-associated defects in complex tissues.

Abstract Foxtail millet is a small diploid C4 crop that possesses superior nutritional properties. It provides a high content of essential amino acids, vitamins, and minerals. In this study, we used CRISPR/Cas9 to simultaneously edit two α-prolamin genes in foxtail millet. Analysis of the derived homozygous lines revealed a decrease in prolamin content. Surprisingly, the content of FAAs such as lysine, taurine, GABA, tryptophan, and sialic acid as well as glucose, fructose, sucrose, and maltose, was significantly increased. Additionally, peak viscosity, final viscosity, trough viscosity, and other parameters showed significant differences, suggesting that the food quality of the gene editing lines may have been altered. The editing lines grew normally, with only a slight decrease in 1000-grain weight compared to the wild type. Overall, we successfully created new germplasm with significantly increased FAAs content through gene editing, providing a valuable reference for the selection of functional foxtail millet varieties.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a highly deadly cancer with limited treatment options. The base excision repair (BER) pathway, crucial for fixing DNA abasic sites, is driven by apurinic/apyrimidinic endonuclease 1 (APE1). While APE1’s redox function has been extensively studied, its endonuclease activity in PDAC homeostasis and therapeutic response remains poorly understood. We created stable, homozygous APE1 endonuclease-reduced PDAC cell lines to examine the effects of impaired BER activity on pancreatic cancer growth, progression, and response to treatment. Methods CRISPR/Cas9-mediated editing was used to introduce an E96A mutation into the Pa03C PDAC cell line, generating three clonal mutant cell lines: E96A B1, E96A B4, E96A G8. APE1 expression and activity were verified in vitro through biochemical assays. Cellular responses to genotoxic stress were examined using cytotoxicity, colony formation, and mtDNA damage assays. Transcriptomic changes were evaluated via RNA sequencing. In vivo tumor growth and metastatic dissemination were studied in orthotopic PDAC mouse models, with and without temozolomide (TMZ) treatment. Results The E96A mutant cell lines exhibited significantly decreased endonuclease activity but had no changes to redox signaling and protein expression. Short-term cytotoxic assays revealed no enhancement in acute sensitivity; however long-term assessment demonstrated a proliferative defect and a vulnerability to genotoxic stress. Transcriptomic analysis revealed that the mutant cell lines maintain a stressed phenotype at baseline, which becomes more pronounced following genotoxic stress. In vivo , E96A mutants had notably lower tumor burden and metastasis at baseline, and the mutation potentiated the effect of the alkylating drug temozolomide, which further inhibited tumor growth and metastasis in a dose-dependent manner. Conclusion We established the first stable human PDAC cell models deficient in APE1 endonuclease activity. Our findings demonstrate that selective impairment of APE1’s DNA repair function expands therapeutic options by lowering the threshold for effective DNA damage validating combination treatments with targeted inhibitors and DNA-damaging agents. Targeting APE1 endonuclease activity represents a promising therapeutic strategy for PDAC, capable of suppressing metastatic spread and enhancing tumor responsiveness to alkylating and other genotoxic therapies.

Background: The coordinated improvement of yield, quality and resistance is a primary goal in rice breeding. Gene editing technology is a novel method for precise multiplex gene improvement. Methods: In this study, we constructed a multiplex CRISPR/Cas9 vector targeting yield-related genes (GS3, OsPIL15, Gn1a), fragrance gene (OsBADH2), and rice blast resistance gene (Pi21) to pyramid traits for enhanced yield, quality, and disease resistance in rice. A tRNA-assisted CRISPR/Cas9 multiplex gene editing vector, M601-OsPIL15/GS3/Gn1a/OsBADH2/Pi21-gRNA, was constructed. Genetic transformation was performed via Agrobacterium-mediated method. Mutation editing efficiency was detected in T0 transgenic plants. Grain length, grain number per panicle, 2-acetyl-1-pyrroline (2-AP) content, and rice blast resistance of homozygous lines were measured in the T2 generations. Results: Effectively edited plants were obtained in the T0 generation. The simultaneous editing efficiency for all five genes reached 9.38%. The individual gene editing efficiencies for Pi21, GS3, OsBADH2, Gn1a, and OsPIL15 were 78%, 63%, 56%, 54%, and 13%, respectively. Five five-gene homozygous edited lines with two genotypes were selected in the T2 generation. Compared with the wild-type (WT), the edited homozygous lines showed increased grain length (2.46%–8.62%), increased grain length-width ratio (3.31%–5.67%), increased grain number per panicle (14.47%–27.11%), a 42–64 folds increase in the fragrant substance 2-AP content, and significantly enhanced rice blast resistance. Meanwhile, there were no significant changes in other agronomic traits. Conclusions: CRISPR/Cas9-mediated multiplex gene editing technology enabled the simultaneous editing of genes related to rice yield, quality, and disease resistance. This provides an effective approach for obtaining new japonica rice germplasm with blast resistance, long grains, and fragrance.

Sorghum (Sorghum bicolor L. Moench) is a vital drought-tolerant crop cultivated in arid and semi-arid regions, serving as both food and fodder. However, under environmental stress, sorghum accumulates cyanogenic glucosides (primarily dhurrin), which hydrolyze into toxic hydrogen cyanide (HCN) upon ingestion, posing lethal risks to livestock. This review examines the biochemical pathways of dhurrin synthesis and HCN release, highlighting key risk factors including drought, frost, and improper grazing management. This study is structured to first elucidate the biochemical and risk factors behind sorghum poisoning, then to evaluate a suite of integrated mitigation strategies—from agroforestry to genetic solutions—and finally to discuss the implementation pathways through education and policy.We evaluate mitigation strategies such as agroforestry integration—using nitrogen-fixing trees (Leucaena leucocephala, Gliricidia sepium) to reduce plant stress and provide alternative fodder—alongside feed management techniques (ensiling, sulfur supplementation). Additionally, we explore genetic solutions (low-dhurrin cultivars developed via CRISPR-Cas9) and microbial/phytoremediation approaches (Pseudomonas fluorescens, Eucalyptus camaldulensis) for cyanide detoxification. Farmer education on risk recognition and safe practices emerges as a critical preventive measure. By synthesizing current research, this paper proposes integrated, sustainable strategies to minimize sorghum poisoning while maintaining agricultural productivity in vulnerable regions.

Abstract Mini zinc finger (MIF) proteins are plant-specific zinc finger-homeodomain (ZF-HD) transcription factors lacking a homeodomain, whose biological functions are critical for normal plant development and the response to environmental stress. Here, CRISPR-Cas9 was used to engineer null alleles of rice OsMIF1 and OsMIF2, and the resulting OsMIF1- and OsMIF2-deficient knockout lines were used to identify the biological roles of OsMIF1 and OsMIF2. The results suggest that OsMIF proteins transcriptionally regulate grain size by controlling the size of epidermal cells and the length and branching of rice panicles. RNA-seq analysis of OsMIF-knockout cells revealed altered expression of genes involved in development, the response to environmental stress and grain size. In addition, 10 protein-interacting partners of OsMIF1 were identified using a yeast two-hybrid screen: these proteins play roles in diverse developmental, hormonal, stress response, and metabolic processes, suggesting that OsMIF1 is effectively a regulatory hub, whose role is to integrate signals as they propagate through rice development- and stress response pathways. The results presented here support the conclusion that OsMIF1 and OsMIF2 are master transcription factors that regulate development throughout the adult plant life cycle and contribute significantly to plant resilience in the presence of environmental stressors.

Abstract Background : While KCNQ1 mutations (I Ks channel α-subunit) are known to cause long QT syndrome (LQTS) presenting with atrial fibrillation (AF), the underlying mechanisms remain incompletely characterized. Methods : We report a novel KCNQ1 c.625T>C (p.Ser209Pro) mutation identified through whole-exome sequencing and Sanger validation in a LQTS pedigree with atypical AF presentation. Utilizing non-invasive urine-derived epithelial cells, we generated integration-free induced pluripotent stem cells (iPSCs) from patients (S209P-iPSC) and established precise homozygous repair of the mutation via CRISPR/Cas9 to create isogenic controls (GC-iPSC), complemented by healthy control iPSCs (CTRL-iPSC). Critically, we developed an atrial-specific differentiation protocol yielding patient-derived atrial cardiomyocytes (aCMs). Patch-clamp and multi-electrode array electrophysiological analyses revealed prolonged action potential duration and delayed repolarization in mutant aCMs—providing the first direct evidence that KCNQ1 dysfunction drives AF susceptibility through impaired atrial repolarization, resolving a key mechanistic gap in channelopathy-associated arrhythmogenesis. Results : Electrophysiological analysis revealed the KCNQ1 c.625T>C mutation induces a tissue-specific channelopathy: patient-derived aCMs exhibited significantly prolonged field potential duration (FPDc) and reduced I Ks current versus controls – indicating distinct atrial-selective repolarization impairment. Crucially, antiarrhythmic testing uncovered paradoxical responses: amiodarone, though clinically used for AF prevention, exacerbated atrial pathology by further prolonging FPDc in dose-dependent fashion and inducing torsade de pointes-like arrhythmias in mutant atrial cells, whereas nadolol normalized FPDc. This mechanistic discordance manifested clinically where amiodarone failed to terminate AF but induced long QT in familial patients – providing the first direct evidence of KCNQ1-mediated atrial vulnerability to proarrhythmic drug effects. Conclusion : The KCNQ1 c.625T>C mutation causes atrial-specific delayed repolarization via I Ks reduction, driving AF in LQTS. We resolve the amiodarone proarrhythmia paradox and establish CRISPR-edited iPSC-atrial myocytes as a transformative platform for precision antiarrhythmic therapy.

Abstract Background Asthenoteratozoospermia, characterized by impaired sperm motility and abnormal morphology, is a major cause of male infertility. However, its genetic basis remains largely unclear in many idiopathic cases. Testis-expressed protein 44 (TEX44) is critical for murine sperm flagellar development, but its role in human asthenoteratozoospermia is remains poorly defined. Methods Whole-exome sequencing (WES) was performed on 535 unrelated infertile men to identify TEX44 variants, followed by Sanger sequencing validation. SWISS-MODEL and IUPred3 were used for TEX44 protein structure and disordered region prediction. A Tex44-knockout (Tex44) mouse model was established via CRISPR/Cas9. Sperm quality was assessed by computer-assisted sperm analysis (CASA). Fertility assays (in vivo fertilization, IVF, ICSI) and ultrastructural observations (TEM, SEM) were conducted to evaluate phenotypic defects. Transcriptome sequencing was used to explore underlying mechanisms. Clinical ICSI outcomes of patients with TEX44 variants were analyzed. Results Eight distinct TEX44 variants (seven missense: c.31G > C, c.236A > G, c.541C > T, c.736T > C, c.781T > C, c.794C > T, c.1156C > T; one frameshift deletion: c.429_432del) were identified in seven patients. Functional prediction and 3D modeling confirmed the pathogenicity of these variants, which correlated with patients' asthenoteratozoospermia phenotypes. Tex44 mice recapitulated the human sperm defects, showing drastically reduced motility and fertilization rates. TEM revealed core ultrastructural abnormalities: disruption of the axonemal 9 + 2 microtubule structure (specifically loss of the 7th peripheral doublet microtubule) and defective mitochondrial sheath assembly.Transcriptome analysis showed downregulated flagellar movement-related genes and upregulated mitochondria-associated genes in Tex44 testes. Notably, ICSI effectively rescued fertility in Tex44 mice and achieved favorable pregnancy outcomes in variant-carrying patients. Conclusion TEX44 variants are novel genetic causes of asthenoteratozoospermia, acting by impairing sperm axoneme integrity and mitochondrial sheath assembly. ICSI is a promising therapeutic strategy for affected individuals, highlighting the translational value of TEX44 as a diagnostic marker and therapeutic target. Genetic mutations in TEX44 further expand the genetic landscape underlying male infertility.

Abstract Purpose Testis-specific TEX family genes are critical for spermatogenesis, but TEX43’s function remains uncharacterized. This study aimed to delineate TEX43’s role in spermatogenesis and fertility using murine models and clinical data. Methods Tex43 expression was analyzed via quantitative reverse transcription-polymerase chain reaction (Q-PCR), immunohistochemistry (IHC), and western blotting. A Tex43 knockout (KO) mouse model was generated using CRISPR/Cas9 (targeting exons 1–3). Testicular histology (hematoxylin-eosin [H&E] staining), sperm parameters (morphology via H&E smears, density via hemocytometer, motility via computer-assisted sperm analysis [CASA]), and fertility (in vivo breeding assays, in vitro fertilization [IVF]) were evaluated. Sperm ultrastructure was assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Whole-exome sequencing (WES) identified TEX43 variants in 146 infertile men with asthenoteratozoospermia. Structural modeling of WT/mutant TEX43 was performed via SWISS-MODEL. Results Tex43 is testis-enriched: mRNA expression initiated at postnatal day 18 (round spermatid stage) and peaked in elongating spermatids; TEX43 localized to sperm flagellar microtubules. Tex43-KO mice showed modestly reduced sperm density (28.6 ± 3.2 vs. 41.2 ± 2.9×10⁶ sperm/ml in WT; P < 0.01) but normal testicular architecture, sperm motility, and fertility (litter size: KO 6.8 ± 0.7 vs. WT 7.2 ± 0.5 pups/litter; P > 0.05). TEM revealed increased flagellar end piece "9 + 2" microtubule disorganization in KO sperm (~ 30% vs. ~5% in WT; P < 0.01). WES identified 9 infertile men with TEX43 variants; 4 with exonic variants (e.g., p.R37Q) achieved live births via intracytoplasmic sperm injection (ICSI). Structural modeling showed p.R37Q disrupted hydrogen bonds critical for microtubule binding. Conclusions TEX43 is minimal impact on murine spermatogenesis and fertility, likely due to genetic redundancy. Human TEX43 variants may exert subtle reproductive effects, requiring validation in larger cohorts with functional studies.

Abstract Gastric cancer (GC) progression is linked to immune escape in the tumor microenvironment, yet the molecules regulating tumor-associated macrophage polarization and CD8⁺ T-cell exhaustion are unclear. This study analyzed TCGA data to examine SULF1 expression and its prognostic role. It used CRISPR/Cas9 and lentiviral methods in GC cells to test proliferation, invasion, and apoptosis, plus co-culture and flow cytometry to assess SULF1’s impact on macrophages and CD8⁺ T-cells. STAT3 signaling was studied via immunoblotting and nuclear translocation assays, and a mouse model tested SULF1’s therapeutic relevance. Results showed SULF1 was up-regulated in GC, tied to advanced stages and poor survival. SULF1 knockdown inhibited GC cell traits, while overexpression boosted them. SULF1 activated macrophage STAT3, promoting M2 polarization and CD8⁺ T-cell dysfunction. In mice, SULF1 silencing reduced tumors and T-cell exhaustion, while supplementation reversed this. Conclusions: GC-secreted SULF1 creates an immunosuppressive microenvironment via STAT3-dependent pathways, and targeting SULF1–STAT3 may improve GC immunity.