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.

The pursuit of extending human healthspan and lifespan has become a central focus in modern biomedical research. Aging is a complex, multifactorial process influenced by genetics, epigenetics, environmental factors, and stochastic molecular events. Traditional approaches, relying on observational studies or single-omic analyses, have provided limited mechanistic insights into the determinants of longevity. Recent advances in multi-omics, genome editing, and artificial intelligence (AI) now offer a transformative framework for predictive and personalized longevity research. In particular, the integration of AI-driven computational modeling, CRISPR-based genome engineering, and comprehensive multi-omic datasets holds the promise of elucidating key molecular drivers of aging and informing targeted interventions.

Abstract Mirusviruses represent a deep evolutionary lineage of DNA viruses, proposed to bridge the viral realms of Duplodnaviria and Varidnaviria. Previously considered to be strictly aquatic viruses infecting unicellular eukaryotes, their full host range and ecological scope have remained enigmatic, thus limiting our understanding of their role in the tree of life. Here, through a global metagenomic survey across 24 terrestrial and aquatic ecosystems, we uncover the hidden diversity and evolutionary breadth of mirusviruses. We reveal that mirusviruses are not confined to aquatic environments but also abundant and diverse in terrestrial biomes, forming novel, deeply-branching phylogenetic lineages. Strikingly, convergent evidence from CRISPR-spacer matches and viral footprints in host genomes suggests that mirusviruses cross the domain boundary, infecting not only eukaryotes but also archaea across ten different phyla. This discovery fundamentally redefines mirusviruses as cross-domain viruses and provides compelling support for the 'mirusvirus origin' hypothesis, which posits their evolutionary origin to predate that of nucleocytoviruses. Our findings place mirusviruses at the nexus of ancient virus-host evolutionary dynamics, suggesting they served as ancestral hubs for cross-domain gene exchange at the dawn of eukaryogenesis.

Abstract SIRPA delivers an anti-phagocytic “don’t-eat-me” signal through its expression on macrophage membrane surface and promotes tumor progression via membrane-independent mechanism. However, current CD47- and SIRPA-targeting agents only disrupt cell-surface inhibitory signaling, highlighting the therapeutic potential of degrading SIRPA. Here, we identified that E3 ubiquitin ligase TRIM2 interacted with SIRPA in vitro. Clinically, elevated TRIM2 expression is associated with prolonged overall and progression-free survival in renal-cell carcinoma (RCC) patients. Mechanistically, TRIM2 catalyzes K48-linked poly-ubiquitination of SIRPA, promoting its proteasomal degradation and reducing SIRPA protein levels. CRISPR/Cas9-mediated deletion of TRIM2 in macrophages upregulated SIRPA, significantly impairing phagocytosis of tumor cells. TRIM2 deficiency also promoted tumor growth by increasing intratumoral infiltration of M2-type macrophage, reducing accumulation of anti-tumor M1-like and antigen-presenting macrophages, and impairing effector CD8 + T-cell recruitment. Importantly, combined TRIM2 overexpression and PD-L1 blockade synergistically enhance anti-tumor immunity. Together, these results suggest that targeting TRIM2 may represent a novel therapeutic strategy against RCC by degrading SIRPA in macrophage and provide a roadmap for clinical application.

In vitro fertilization (IVF) has long been a cornerstone of assisted reproductive technologies (ART) in animals, widely used in livestock breeding, endangered species conservation, and biomedical research. Traditional IVF techniques, while effective, often rely on trial-and-error protocols and are influenced by various biological and environmental factors. Recent innovations in biotechnology are revolutionizing this field. The integration of CRISPR-based genome editing, artificial intelligence (AI), and multi-omics technologies is propelling animal IVF into a new era of precision, efficiency, and predictability. In this review, the potential and recent researches of IVF in animals advancing with CRISPR, AI and omics were discussed before including future directions of this valuable field.

We have previously demonstrated that heterozygous (HET) female mice lacking one copy of the X-linked gene Nexmif display autistic-like phenotypes, memory impairments, and deficits in synapse and neuron morphology. Due to random X Chromosome Inactivation (XCI), the HET mouse brain contains two populations of neurons: NEXMIF-expressing cells (wildtype, WT) and NEXMIF-lacking cells (knockout, KO). Interestingly, because KO cells contain a normal WT copy of Nexmif on the inactivated X chromosome (Xi), we wondered whether the silenced Xi- Nexmif could be reactivated to restore NEXMIF expression in neurons as a strategy to correct this mosaic deficiency in HET mice. To this end, we first tested pharmacological inhibition of XCI maintenance and found that intracortical administration of the DNA methylation inhibitor 5-aza-2’-deoxycytidine combined with resveratrol (Aza+Resveratrol) increased NEXMIF expression in HET mice. Using a gene-specific approach, we developed a NEXMIF -targeted CRISPR activation (CRISPRa) system and found that it selectively increases NEXMIF transcription in human female cells and in vivo in WT female mice with minimal off-target effects. Importantly, CRISPRa restored NEXMIF expression in the KO neurons of HET primary cultures, effectively correcting XCI-driven mosaicism. These findings demonstrate that pharmacological- and especially CRISPRa-mediated reactivation of the Xi may serve as a strategy for the reversal of neuronal and behavioral impairments in Nexmif HET conditions.

ABSTRACT High-risk endometrial cancers (EC), such as uterine carcinosarcomas (UCS) and serous endometrial intraepithelial carcinoma (SEIC), are characterized by frequent mutations in tumor suppressor genes (TSGs) and poor clinical outcomes. Traditional genetically engineered mouse models are limited in flexibility and scalability to study the cooperative effects of multiple TSG alterations. Here, we use a multiplexed CRISPR/Cas9-based approach to simultaneously edit the top ten TSGs commonly mutated in high-risk EC directly in the mouse endometrium via intrauterine electroporation. Using rolling circle amplification (RCA) and next-generation sequencing, we demonstrate that this method induces targeted gene editing in a mosaic manner, mimicking tumor heterogeneity. We demonstrate that this approach generates histologically and molecularly faithful models of SEIC and UCS. Importantly, some edited tissues remained histologically normal, emphasizing the complex multistep nature of endometrial tumorigenesis. These CRISPR/Cas9-generated murine models serve as robust platforms to dissect the molecular underpinnings of high-risk endometrial cancer and to accelerate preclinical evaluation of novel therapeutic strategies.

Core members of the fungal root microbiota include pathogens capable of colonizing multiple hosts, yet the underlying genetic determinants remain unknown. We report that Plectosphaerella cucumerina is a core member of the Arabidopsis thaliana root microbiota displaying high pathogenic potential and multi-host colonization capabilities. Establishment of a Plectosphaerella reference culture collection, followed by whole-genome sequencing of 72 strains reveals subtle phenotypic and genotypic variation that associate with fungal phylogeny, but not host plant identity. Transcriptome profiling of a model P. cucumerina isolate in roots of multiple hosts identifies core and host-specific fungal processes linked to carbon catabolism and root cell wall deconstruction of the hosts. A fungal gene encoding a candidate β-1,3-glucanase (GH64) was identified as a key genetic factor driving infection and disease in plants that diverged 110 million years ago. The gene is enriched in plant-colonizing fungi and consistently functions as a disease determinant in the root pathogen Colletotrichum incanum . We conclude that diverse and tunable fungal repertoires of carbohydrate-active enzymes act as disease determinants and drive multi-host compatibility belowground.

Abstract Microbial keratitis (MK) is a major global cause of blindness. Yet, treatment is still heavily dependent on antimicrobials with limited options for immunomodulators - despite the critical role of dysregulated immune responses in disease pathogenesis. This gap reflects a critical unmet clinical need and is compounded by the lack of model systems capable of real-time high-resolution immune dynamics analysis. To address this, we developed a zebrafish larvae MK model utilising transgenic zebrafish lines with fluorescently labelled neutrophils, macrophages and basal epithelial cells. Corneal injury triggered rapid immune cell recruitment which was amplified by exposure to pro-inflammatory mediators such as N-formylmethionine-leucyl-phenylalanine (fMLF) and leukotriene B4 (LTB4). Infection with live bacteria induced robust, sustained neutrophil and macrophage recruitment, marked by increased neutrophil speed and migratory distance. This model enables dynamic in-vivo visualization of immune cell dynamics, offering a powerful and scalable platform to accelerate the discovery and screening of novel immunomodulators for MK.

The increasing demand for ethically acceptable, economically viable, and translationally relevant animal models in biomedical research positions Danio rerio (zebrafish) as a prominent alternative to traditional rodent systems. This review provides an integrated analysis of zebrafish biology and delineates their expanding applications in pharmacological investigations and toxicological evaluations. Emphasis is placed on genetic homology with humans, optical transparency during embryogenesis, and suitability for high-throughput screening, which collectively support the model’s relevance in contemporary biomedical studies. The historical progression of zebrafish usage is outlined, and critical biological features, such as developmental kinetics, sexual dimorphism, and organogenesis are described to contextualize their utility in disease modeling. Zebrafish are examined for their capacity to assess acute, chronic, and specialized toxicity endpoints, including neurotoxicity, hepatotoxicity, and endocrine disruption. Their roles in investigating inflammation, metabolic disorders, neurodegeneration, cancer, and infectious diseases are also reviewed. Technological advancements, including CRISPR/Cas9-mediated gene editing and the development of transgenic lines, are discussed alongside innovations in imaging and screening methodologies. Regulatory frameworks, as well as compliance with Good Laboratory Practices (GLP), are addressed. The review concludes by evaluating the potential of zebrafish in precision medicine and their capacity to enhance early-phase drug discovery through scalable, cost-effective, and biologically relevant approaches.

In vitro air-liquid interface culture of airway epithelial cells is used as a model system to study respiratory diseases. This culture system not only overcomes the need for animal models or continuous biopsies from individuals but also enables studies of pathophysiology associated with the disease in a patient background. Human airway basal cells serve as progenitor cells for a functional pseudostratified airway epithelium composed mainly of multiciliated and secretory cells. However, due to the limited ability of basal cells to proliferate and differentiate, the long-term use of primary material in culture is restricted. This challenges research that requires genome editing. Here, we describe airway stem cells from nasal and bronchial origin immortalized by hTERT overexpression followed by polyclonal expansion. We demonstrate that this diverse panel of cell lines shows differentiation patterns similar to primary stem cells and can be used for lentiviral and CRISPR/Cas9 genome editing. These cell lines and optimized protocols facilitate airway biology research and disease phenotyping.

ABSTRACT Heterobifunctional proteolysis-targeting chimeras (PROTACs) have emerged as a powerful strategy to degrade disease-relevant proteins, enabling targeting of previously “undruggable” proteins. Current degrader molecules primarily target cytosolic substrates, yet nearly one-third of the proteome resides in or transits the endoplasmic reticulum (ER), including receptors, secreted factors, and biosynthetic enzymes with high therapeutic relevance. Whether ER-localized proteins can be broadly targeted for induced degradation remains an open question. To address this gap, we employed a panel of fluorescent reporter cell lines and used the dTAG chemical-genetic system to recruit cytosolic E3 ligases. While lumenal substrates segregated from the cytosol were resistant to degradation, recruitment of cytosolic ligases effectively degraded ER membrane proteins across multiple topologies and with post-translational modifications. CRISPR genetic screens revealed that the induced degradation required the expected cullin RING ligase complexes but surprisingly bypassed ER-associated degradation (ERAD) machinery, with the exception of the AAA ATPase VCP. Mechanistic studies demonstrated that substrate ubiquitination was essential for VCP binding, and cleavage of ubiquitin chains released VCP, suggesting a model in which VCP directly extracts substrates independent of a dislocation apparatus. Extending this strategy to an endogenous substrate, we synthesized an HMGCR ERAD-TAC by linking atorvastatin to a cereblon E3 ligase recruiter and found that HMGCR degradation was likewise VCP-dependent. Together, these findings demonstrate that ER membrane proteins are generally susceptible to induced degradation via cytosolic ligase recruitment, uncovering a VCP-centered mechanism that operates independently of membrane-embedded ERAD machinery. This work establishes foundational principles for extending targeted protein degradation to the early secretory pathway. SIGNIFICANCE STATEMENT Targeted protein degradation has transformed drug discovery. Nearly one-third of the proteome reside in or transit the endoplasmic reticulum (ER), a compartment rich in therapeutically relevant but structurally complex targets. Whether these ER proteins can be broadly degraded using PROTACs has remained unknown. Here, we define the minimal requirements for degrading ER membrane proteins by recruiting cytosolic E3 ligases. Using chemical-genetic tools, genetic screens, and a statin-based degrader, we show that ubiquitination engages the VCP extraction machinery, enabling degradation of diverse ER membrane proteins independent of canonical ER-associated degradation components. These findings reveal a ubiquitin-driven route for membrane protein turnover, expand the landscape of druggable ER proteins, and establish principles for designing degraders operating in the early secretory pathway.

Glioblastoma (GBM) is the most common primary malignant brain tumour in adults with dismal survival rates, and current therapies, including most immunotherapies, are not efficacious due to the highly immunosuppressive microenvironment. Studies in other solid cancers report that impairment of the integrin effector pathway involving focal adhesion kinase (FAK) can promote anti-tumour immune responses. Therefore, we set out to address whether, and if so how, suppressing FAK function may influence GBM by using both tumour cell-specific FAK gene deletion and systemic delivery of a clinically relevant FAK kinase inhibitor (FAKi) VS-4718 in an orthotopic murine stem cell model of GBM. We found that treatment with the FAKi, but not tumour cell-specific FAK gene deletion, resulted in GBM clearance and improved survival. This was dependent on adaptive immunity, and tumour-infiltrating T cells in FAKi-treated tumours displayed increased cytotoxic potential and reduced exhaustion. We also found a significant reduction in immuno-suppressive peripherally-derived macrophages and FAKi treatment caused sequestration of inflammatory monocytes within the bone marrow, resulting in impaired monocyte trafficking to tumours as judged by adoptive transfer. This is due to suppression of key adhesion and migration signalling through α4β1 integrin and CX3CR1 in peripheral monocytes. Our work here describes a previously unidentified role for FAK in trafficking of peripheral suppressive macrophages to GBM tumours, reducing T cell exhaustion and promoting anti-tumour immunity. This highlights a new way in which systemic FAK inhibitors can be used to provide a beneficial immune modulatory strategy for the treatment of GBM.

Re-evaluating existing clinical compounds can uncover previously unrecognized mechanisms that reshape a drug's therapeutic potential. The small molecule Procaspase-Activating Compound 1 (PAC-1) entered oncology testing as a proposed activator of caspase-driven apoptosis. Here, we show that PAC-1-driven cytotoxicity occurs in the absence of executioner caspase expression, demonstrating that its anti-cancer activity occurs via an alternative mechanism. We provide genetic, biochemical, and biophysical evidence demonstrating that PAC-1 functions as a highly selective iron chelator that eliminates cancer cells by disrupting iron homeostasis. Unexpectedly, we discovered that expression of the key chemotherapy-resistance pump MDR1 confers marked hypersensitivity to PAC-1 treatment. While PAC-1 is only weakly effluxed by MDR1 under basal conditions, this process is potentiated when PAC-1 is bound to iron. Consequently, PAC-1 induces progressive iron depletion and selective cytotoxicity in otherwise drug-resistant MDR1-expressing cancer cells. Together, these findings redefine PAC-1's mechanism-of-action and establish a framework for exploiting multidrug resistance as a therapeutic vulnerability through targeted iron starvation.

Host-pathogen interactions are shaped by cellular restriction factors that direct antiviral defenses. We built the first ovine genome-wide CRISPR knockout library in sheep testis (OA3.Ts) cells, targeting all protein-coding genes. Using this platform, we identified PEX11B, a peroxisomal membrane regulatory protein, as a strong restriction factor against orf virus (ORFV) infection. Removing PEX11B increased viral susceptibility and triggered severe cytopathic effects with membrane fusion and syncytia formation. Mechanistic studies showed that PEX11B knockout harmed peroxisomal integrity and disrupted lipid metabolism. This led to greater plasma membrane fluidity, creating a proviral environment that allowed more viral entry and replication. These results reveal a new antiviral function for PEX11B in blocking viral infection and underscore the importance of peroxisomal regulation in host-virus interactions.