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.

Development of the next generation of chimeric antigen receptor (CAR) T-cells requires assessment in systems that better recapitulate the suppressive tumour microenvironment of solid tumours. CRISPR-Cas9 knock-in of promoter-less homology directed repair templates (HDRT) into the T-cell receptor locus has been shown to result in physiological expression of CARs with improved tumour control. We initially compared the use of dsDNA and adenovirus associated virus (AAV) HDRTs in mouse T cells. We have subsequently developed an optimised method for AAV transduction resulting in high editing efficiencies with minimal toxicity. In contrast with our experience of retroviral transduction of mouse T cells, our CRISPR/Cas9 AAV transduction method results in sustained CAR expression and T cell expansion in vitro as well as in vivo persistence. This approach allows for pre-clinical assessment of individual and libraries of CAR constructs in relevant immune-competent mouse models.

CRISPR-mediated genome editing of the central nervous system (CNS) has the potential to revolutionize the treatment of neurological disorders, including neurodegenerative disorders such as Huntington’s disease (HD). However, the development of CRISPR therapeutics for the CNS has been hindered by challenges associated with delivery, specifically the lack of a clinically compatible, non-viral delivery technology facilitating genome editing of neurons in vivo . For most indications, two key obstacles must be overcome before therapeutic genome editing of the brain is feasible: non-toxic intracellular delivery of CRISPR cargo into neurons and establishment of strategies enabling targeted brain regions to be edited efficiently. While viral vectors have shown promise in pre-clinical models, non-viral approaches present distinct advantages: ease of manufacture as well as the transient presence of CRISPR machinery, which tempers risks of genotoxicity and immunogenicity. Peptide-enabled ribonucleoprotein (RNP) delivery of CRISPR (PERC) has emerged as a promising non-viral delivery strategy for CRISPR enzymes with initial use in primary human immune cells. In this study, we report the development of Neuro-PERC, a streamlined and optimized approach for in vivo editing of mammalian neurons. Administration of Neuro-PERC reagents via convection-enhanced delivery (CED) mediated efficient and well-tolerated neuronal genome editing. Neuro-PERC enabled robust neuronal editing in the brain of both small and large animal reporter models, and increased survival in a severe murine model of Huntington’s disease. These results establish CED-administered Neuro-PERC as a candidate delivery technology to hasten clinical translation of CRISPR-based therapies for diseases of the CNS. Summary Neuro-PERC, a peptide-mediated CRISPR enzyme delivery technology, enables efficient in vivo mammalian neuronal editing in the brain of mice and pigs, extending survival in a murine model of Huntington’s disease when administered via convection-enhanced delivery (CED).

Deserts cover a third of the world's surface, supporting unique biomes and ecosystem services. Yet, we lack a comprehensive assessment of what defines and drives the microbial communities that dominate life in these regions. Here, we conducted a standardized field survey in contrasting cold, hot, and polar deserts across the seven continents, and observed geographically distant deserts share similar structure, function, and activities. Desert communities are dominated by genomically streamlined Actinobacteriota and Chloroflexota, and compared with non-desert soils, are significantly enriched with stress tolerance genes, mobile genetic elements, and antiviral strategies, revealing previously unknown ecological and evolutionary dynamics. Metabolically, these communities exhibit reduced capacity for carbohydrate and protein degradation, and instead are enriched for chemosynthetic carbon fixation, continuous energy harvesting using atmospheric trace gases and sunlight, and energy reserve biosynthesis. All sampled soils mediated respiration, trace gas oxidation, and carbon fixation, with detectable activity even in hyper-arid Atacama and Antarctic soils at the margins of life. Driver analyses identified aridity as the primary overriding driver of the microbial communities and biogeochemical activities. Collectively, these findings suggest that aridity selects for metabolically self-sufficient taxa capable of continuously meeting energy and carbon needs independently of vegetation-derived inputs, while enduring physicochemical stressors and potentially elevated viral pressure. These new insights are integral to forecast the future of soils amid increasing desertification.

Summary Despite their recognized role in biology, a majority of the ∼100,000 lncRNA genes remain functionally uncharacterized. In a recent study ( Liang WW et al ., Transcriptome-scale RNA-targeting CRISPR screens reveal essential lncRNAs in human cells, Cell, 2024) , Liang et al. utilized the RNA nuclease Cas13d to perturb ∼6,200 lncRNAs in fitness screens across five cell lines - thereby identifying 778 lncRNAs with broad or context-specific essentiality. However, previous screens reported a lower proportion of essential lncRNAs. To investigate this discrepancy, we re-analysed Liang et al.’s data and found that 68.1% of gRNAs causing fitness defects have off-targets in essential protein-coding genes. This caused numerous false-positive hits, particularly among lncRNAs classified as broadly essential. Off-target effects also compromise the study’s validation efforts, including experiments combining single-cell transcriptomics and lncRNA-perturbations, which confirm the downregulation of off-target protein-coding genes identified in our analyses. The large number of false-positive hits reported by Liang et al. undermines the study’s biological conclusions and endangers future research building on these data, if not considered.

Archaea, the third domain of life, play critical roles in global biogeochemical cycles. However, their virosphere, particularly the proviruses which integrated into host genomes, remains largely unexplored. To systematically reveal the landscape of archaeal proviruses, we conducted large-scale mining of public and in-house genomic datasets spanning all presently known archaeal phyla. We identified 9,697 archaeal proviruses across 19 archaeal phyla and 366 families, which clustered into 9,123 viral operational taxonomic units (vOTUs). Among these, 97.2% represent novel viruses, and 81.3% could not be classified at the family level, substantially expanding the known diversity of archaeal viruses. Host range analysis revealed that many proviruses exhibit broad infectivity across archaeal lineages, with some even capable of cross-domain infection. Genomic analysis identified 178 distinct types of antiviral systems in archaeal hosts, encompassing multiple CRISPR-Cas variants and restriction-modification (RM) systems. Meanwhile, we detected 747 anti-defense genes encoded by 710 proviruses, such as anti-CRISPR and anti-RM, directly corroborating the ongoing evolutionary arms race between archaeal hosts and their viruses. Additionally, we identified 532 auxiliary metabolic genes (AMGs) within archaeal proviruses that are involved in key processes including carbon, nitrogen, and sulfur metabolism, indicating their potential to reprogram host metabolic pathways and thereby influence biogeochemical cycling. This study establishes a systematic global genomic atlas of archaeal proviruses, advancing our understanding of their distribution and diversity while laying the groundwork for future investigations into how AMG-mediated processes influence archaeal metabolism and ecosystem functions.

Inositol pyrophosphates (PP-InsPs) are key nutrient messengers in plants, but their protein receptors remain poorly defined. Using a systems-level affinity screen with biotinylated InsP₆, InsP₇, and InsP₈ in Arabidopsis thaliana , we identify multiple conserved PP-InsP-interacting complexes involved in mRNA metabolism, translation, and cell signaling, including the nuclear α-subunits of casein kinase II (CK2). The CK2 subunit AtCKA1 associates with the PP-InsP kinase AtVIH2, and its 1.9 Å crystal structure with InsP 6 reveals two conserved PP-InsP binding sites located in the N-and C-terminal lobes. AtCKA1 binds InsP 6 , InsP 7 , and InsP 8 with micromolar affinity. Mutation of both binding sites in the AtCKA 6xmut mutant abolishes PP-InsP binding in vitro. AtCKA 6xmut partially rescues the flowering phenotype of ck2a1/2/3 mutants, and equivalent mutations inactivate the yeast orthologs ScCka1 and ScCka2. InsP 6 competitively inhibits phosphorylation of canonical CK2 substrates by occupying a basic substrate-binding groove. Although incorporating β-subunits strongly enhances the phosphorylation of substrates by the AtCK2 holoenzyme, ck2b1/2/3/4 mutants exhibit only mild growth defects in Arabidopsis. In Marchantia , loss of the single ck2a gene severely impairs growth, whereas deletion of the β subunit has no effect. Together, our findings suggest that InsP 6 /PP-InsPs modulate the activity of the isolated CK2 α-subunit by regulating access to its substrate-binding site.

Tardigrades, commonly known as water bears or moss piglets, are microscopic extremophiles famed for their resilience to harsh environments, including extreme temperatures, radiation, and desiccation. Beyond their unique survival traits, recent discoveries of their symbiotic microbial partners open exciting avenues for biotechnological innovation. This review explores the untapped potential of tardigrades and their symbionts as novel sources for next-generation superfoods, cosmetics and pharmaceuticals. Leveraging CRISPR gene-editing technology, we discuss strategies to enhance their nutritional profiles, stress tolerance, and metabolic pathways for sustainable food production. Integrating extremophile biology with cutting-edge genome engineering could revolutionize food systems by introducing robust, nutrient-dense, and environmentally resilient bioresources.

ABSTRACT During fertilization, haploid gametes combine to form a zygote. The male (sperm) and female (oocyte) gametes contribute a similar amount of DNA, but the oocyte contributes nearly all the cytoplasm. Oocytes are loaded with maternal mRNAs thought to be essential for embryonic patterning after fertilization. A conserved suite of RNA-binding proteins (RBPs) regulates the spatiotemporal translation and stability of maternal mRNAs. POS-1 is a CCCH-type tandem zinc finger RBP expressed in fertilized Caenorhabditis elegans zygotes from maternally supplied mRNA. POS-1 accumulates in the posterior of the embryo where it promotes posterior cell fate. Here, we show that the pos-1 3ʹ untranslated region (UTR) is essential for POS-1 patterning and contributes to maximal reproductive fecundity. We engineered a pos-1 mutant where most of the endogenous pos-1 3ʹUTR was removed using CRISPR genome editing. Our results show that the 3ʹUTR represses POS-1 expression in the maternal germline but increases POS-1 protein levels in embryos after fertilization. In a wild-type background, POS-1 repression via the 3ʹUTR has little impact on fertility. In a sensitized background, the deletion mutant has a complex pleiotropic phenotype where most adult homozygous progeny lack either one or both gonad arms. Most phenotypes become more penetrant at elevated temperature. Together, our results support an emerging model where the 3ʹUTRs of maternal transcripts, rather than being essential, contribute to reproductive robustness during stress.

ABSTRACT Cells must continuously sense and respond to environmental changes by translating physical and chemical cues into intracellular signals. However, systematic discovery of genes governing these sensory processes has been limited by the transient nature of signaling events and the low throughput of measurement assays. Here, we present CaRPOOL, a pooled, high-throughput genetic screening platform that integrates the calcium-activity recorder CaMPARI2 with CRISPR interference (CRISPRi), enabling stable capture of transient calcium signals for genome-scale functional screening. Using osmomechanical stimulation as a model, we demonstrate that CaMPARI2 photoconversion faithfully reports stimulus-dependent calcium responses and supports pooled fluorescence-activated cell sorting (FACS)-based screening. A CRISPRi library targeting membrane-associated genes identified both known and previously uncharacterized regulators of mechanotransduction, including the chemokine receptor CCR7. Mechanistic analyses revealed that CCR7 promotes osmomechanical calcium signaling through a PIEZO1-dependent Gαs-cAMP-PKA pathway, establishing it as a mechanosensitive GPCR. Notably, osmotic stress upregulated CCR7 expression in immune cells and enhanced mechanical responsiveness, suggesting a role in immunomechanical adaptation. Together, these findings introduce a broadly applicable platform for high-throughput discovery of genes controlling dynamic signaling responses and reveals a GPCR-ion channel crosstalk mechanism in mechanotransduction with potential implications for immune cell mechanoadaptation.

ERCC6L2 disease is a recessive bone marrow failure (BMF) syndrome caused by mutations in the SNF2-like putative DNA helicase ERCC6L2. While implicated in DNA replication, double strand break (DSB) repair via non-homologous end joining (NHEJ), and interstrand crosslink (ICL) repair, how ERCC6L2 supports haematopoietic longevity remains unclear. Investigating this in vivo , we find that an Ercc6l2- deficient haematopoietic stem and progenitor cell (HSPC) compartment in mice is unexpectedly resilient. Ercc6l2 loss was also tolerated in mice co-deficient for endogenous formaldehyde detoxification, which precipitates early-onset BMF in models of Fanconi anaemia. Instead, Ercc6l2 -deficient mice display a mild immunodeficiency, arising from defects in immunoglobulin class-switch recombination (CSR), that synergise with shieldin-deficiency, implicating ERCC6L2 and shieldin in distinct repair mechanisms. Furthermore, we demonstrate that ERRC6L2 stimulates chromosome fusions in the context of staggered, but not blunt dysfunctional telomeres. We reconcile ERCC6L2’s NHEJ function through proteomic elucidation of its endogenous interactome and AlphaFold structural modelling to reveal a complex formed of ERCC6L2 and KU that is bridged by the NHEJ accessory factor MRI/CYREN. Consequently, ERCC6L2-MRI inter-dependence characterises CSR. Together, our findings implicate the ERCC6L2-MRI complex as a KU-regulatory DNA translocase coordinating classical-NHEJ at staggered-end DSBs. We suggest that similar staggered-end breaks represent the pathological substrates driving haematopoietic failure in ERCC6L2 disease.

Abstract Common wheat ( Triticum aestivum L.) is a staple food crop for humans, yet it primarily accumulates the non-provitamin A carotenoid lutein and exhibits limited natural variation in provitamin A β-carotene among its various accessions. This characteristic necessitates the development of alternative strategies for provitamin A biofortification. To address this challenge, we targeted key control points in the carotenoid biosynthetic pathway using the CRISPR-Cas9 system in a wheat cultivar Fielder. Specifically, we knocked out the gene encoding lycopene ε-cyclase (LCYE), an enzyme that acts as a gatekeeper opposing the production of β-branch carotenoids. This genetic modification resulted in a significant increase in β-carotene levels in the endosperms at 30 DPA of triple homozygous transgene-free mutant lines revealed by biochemical profiling, an approximate 34.5% enhancement for β-carotene, 125.4% for zeaxanthin, 73.8% for violaxanthin, and 186.5% for antheraxanthin compared to the wild-type control. Despite the drastic reduction in lutein levels, the TaLcye mutations did not significantly impair wheat yield and the mutant lines exhibited elevated levels of amylose and soluble sugar. Additionally, the seed coats and endosperms of the triple homozygous transgene-free mutant lines exhibited an orange-yellow hue. In conclusion, we have successfully developed novel carotenoids biofortified wheat lines through gene-editing approach. Our findings demonstrate the potential of gene editing to significantly enhance the nutritional profile of commercial wheat by increasing carotenoid content, thereby addressing micronutrient deficiencies in modern diets.

Abstract Background: Liver Fibrosis represents a significant global health burden as a chronic progressive disease. Emerging evidence has established a critical link between macrophage extracellular traps (METs) and liver fibrosis; however, the precise triggers of MET formationand their mechanistic contributions to fibrosis remain poorly understood. Substantial evidence indicates that interleukin-25 (IL-25) potently regulates macrophage metabolism and fibrotic progression. Methods: A carbon tetrachloride (CCl₄)-induced mouse model of liver fibrosis spanning distinct stages (2 to 8 weeks) was established. In vitro studies utilized co-culture systems and conditioned medium treatment to assess MET-mediated activation of hepatic stellate cells (HSCs). To elucidate the specific role of IL-25, hepatocyte-specific IL-25 knockout (IL-25CKO) mice were generated using CRISPR/Cas9 technology and subjected to the fibrosis model. Mechanistic investigations involved stimulating RAW 264.7 macrophages with IL-25 and specific inhibitors. Techniques such as scanning/transmission electron microscopy, immunofluorescence, Western blot and ELISA were employed to analyze MET formation, ROS production, lysosomal activation, mitophagy, and related signaling pathways. Results: Fibrotic livers exhibited strong early co-localization of IL-25 with hepatocytes, which preceded MET formation. This demonstrates that early production of IL-25 by hepatocytes acts as a triggering factor for MET formation. In vitro co-culture systems revealed MET-mediated activation of HSCs, while mechanistic studies showed that IL-25 promotes IL-17RB receptor activation, triggering downstream reactive oxygen species (ROS) bursts, lysosomal activation, and MET formation. Conclusions: This work identifies a pathogenic hepatocyte–macrophage–HSC axis and supports IL-25 blockade as a promising clinical strategy for liver fibrosis.

The human gut microbiome, composed of trillions of microorganisms including bacteria, viruses, fungi, and archaea, plays a critical role in health, metabolism, immunity, and disease susceptibility. Over the last decade, the complexity and dynamism of the gut microbiota have been increasingly appreciated, revealing intricate interactions with host physiology that influence conditions ranging from obesity, diabetes, and cardiovascular disease to neurodegenerative disorders and cancer. Understanding and manipulating this microbial ecosystem requires integrative approaches that combine advanced molecular techniques, computational analytics, and precise genetic interventions. Recent advances in CRISPR genome editing, high-throughput omics technologies, and artificial intelligence (AI) have collectively transformed gut microbiology research, opening new avenues for predictive modeling, therapeutic interventions, and personalized medicine. This review gathers recent researches in CRISPR, AI and omics in the field of gut microbiology and further discussed future outlooks and perspectives.

Haemoglobinopathies, including β-thalassaemia and sickle cell disease (SCD), are among the most common monogenic disorders worldwide and remain major causes of morbidity and early mortality. Historically, management of these life-altering diseases has relied on supportive treatment and symptom management and although these treatments reduce symptoms and ease disease burden, they do not correct the underlying genetic defect. Allogenic haematopoietic stem cell transplantation (HSCT) has been the only established curative option; however, it comes with substantial risks that significantly restrict its applicability. Over the past two decades, haematopoietic stem cell (HSC) gene therapy for haemoglobinopathies has rapidly progressed from experimental proof-of-concept to approved therapies. Lentiviral gene addition approaches have demonstrated durable expression of functional β-like globin transgenes, achieving transfusion independence in β-thalassaemia patients and significant reductions in vaso-occlusive events in SCD patients. Alternative therapeutic approaches to promote HbF expression have proved to be highly successful. Gene silencing strategies targeting BCL11A have been successful clinically and more recently, gene editing technologies such as CRISPR/Cas9, have enabled precise disruption of regulatory elements controlling γ-globin repression leading to the approval of the first CRISPR-based therapy for SCD and β-thalassaemia. Emerging base-editing technologies promise even more precise genetic modification and are advancing through clinical evaluation. Despite these advances, access to gene therapy remains restricted due to the need for highly specialised manufacturing, toxic myeloablative conditioning regimes, and high treatment costs. Ongoing improvements and adaptations in these areas are essential to ensure that gene therapies fulfil their potential as accessible, curative treatments for patients suffering from haemoglobinopathies worldwide.

Micro-satellite repeat expansion of the 5’ GGGGCC 3’ sequence in the C9orf72 gene is the most common monogenic form of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) translated from the mutant allele can be detected in postmortem brains of afflicted individuals. The arginine containing peptides, poly-PR and poly-GR, are particularly noxious to cells. Both have been shown to undergo cell-cell transmission, but the underlying mechanisms are not understood. We found rapid internalization and nucleolar localization of bath-applied hemagglutinin (HA) tagged poly-PR with twenty repeats (HA-PR 20 ) in cell lines and neurons. Small molecule and RNAi approaches implicated a temperature-dependent, fluid phase endocytosis mechanism in HA-PR 20 uptake. We sought to identify DPR-related cell surface uptake factors using a high-resolution proximity labeling technique developed in the MacMillan group, termed µMap. DPR-iridium conjugates identified candidate cell-surface proteins which were interrogated in an RNAi screen. Focusing on our strongest candidate, chondroitin sulfate proteoglycan 4 (CSPG4), we showed that cellular uptake of HA-PR 20 is blocked by inhibition of glycosaminoglycan chain synthesis (using drugs or RNAi) and knockdown or ablation of CSPG4 (using RNAi or CRISPR editing). Reduction of CSPG4 protected PR 20 -induced neuronal toxicity. We used a dual reporter system to interrogate in vitro neuron-to-neuron transmission of PR 50 and found that PR 50 synthesized by one neuron readily spread to neighboring neurons. Transmission was significantly reduced when CSPG4 was knocked down. These results suggest CSPG4 is an important factor in poly-PR internalization and transmission and therefore may be a therapeutic target to slow DPR transmission and disease progression. Significance Statement A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common monogenic form of ALS/FTD. This expansion leads to dipeptide repeat protein (DPR) production through non-canonical translation of repeat-containing RNA. DPRs have been shown to transmit between cells, but how this occurs is not well understood. We identified the cell surface protein chondroitin sulfate proteoglycan 4 (CSPG4) as a mediator of the uptake and intercellular spread of toxic arginine-rich DPRs. Targeting CSPG4 may provide a strategy to block DPR transmission and slow disease progression.

Acoziborole is a safe, single dose, oral therapy, for treatment of both early and late-stage sleeping sickness, a deadly disease caused by African trypanosomes. Other benzoxaboroles show efficacy against other trypanosomatids, apicomplexans, fungi, bacteria, and viruses. Acoziborole targets the trypanosome pre-mRNA processing endonuclease, cleavage and polyadenylation specificity factor 3 (CPSF3), and triggers CPSF3 degradation, but it remains unclear whether additional mechanisms contribute to efficacy. We used oligo targeting for site saturation mutagenesis of the native CPSF3 gene. Among >1,500 edits around the putative drug binding site, only Asn 232 His edits conferred moderate resistance to acoziborole. Using a novel combinatorial oligo targeting method we edited multiple sites simultaneously, including sites that differ in human CPSF3, and found that an Asn 232 His, Tyr 383 Phe, Asn 448 Gln triple-mutant strain was >40-fold resistant to acoziborole. We used gene tagging to show that all three edits were on the same allele, and to show that triple-mutant CPSF3 was highly resistant to rapid acoziborole and proteasome-dependent degradation. Computational modelling revealed how the combinatorial mutations can disrupt acoziborole – CPSF3 interactions by introducing steric clash and by disrupting hydrophobic and water-mediated interactions. We conclude that acoziborole safety and efficacy can be explained by selective affinity for, and rapid turnover of, trypanosome CPSF3. Author Summary Diagnosis and treatment options, previously limited for sleeping sickness, have been transformed in recent years. Acoziborole, for example, is a new, safe, single dose, oral therapy for the treatment of this deadly disease. This drug can also be used without the need for cumbersome disease-stage diagnosis. Additional boron-based drugs also show great promise against a whole range of other infectious diseases. Acoziborole targets an RNA processing enzyme in African trypanosomes, and triggers its degradation, but human cells express a similar enzyme, and alternative trypanosomal targets have also been suggested. Insights into how a drug interacts with its target can help to understand selective action against a pathogen, and to predict resistance, an ever-present threat for many drugs. We used a precision gene editing method to change the target protein in trypanosomes, editing single sites or multiple sites simultaneously. A novel triple-mutant was found to be both highly resistant to acoziborole and highly resistant to rapid degradation. Using computational models, we were able to explain how multiple mutations interfered with acoziborole binding to its target. The findings show how selective binding of a specific parasite enzyme makes acoziborole such a safe and effective drug.

ABSTRACT Traditional taxonomies classify ferroptosis and apoptosis as distinct forms of regulated cell death. Here, we challenge this view by defining and validating a previously unrecognized neuronal death entity in traumatic brain injury (TBI), which we term “Ferrapoptosis”. This hybrid mode of death is characterized by the coexistence of ferroptotic and apoptotic molecular and ultrastructural features within the same neuron— ultrastructural alterations that cannot be fully characterized by any known cell death modalities. By integrating single-cell transcriptomics with multi-layered functional assays, we show that Ferrapoptosis dominates in severe injury and the acute phase, and is critically driven by mitochondrial oxidative stress. Over time, its predominance is gradually replaced by death modes in which either ferroptosis or apoptosis alone becomes the major pathway. Genome-wide CRISPR screening further identifies Smg7 as a key regulator that synchronously activates both death programs to drive the hybrid phenotype, whereas genetic or viral inhibition of Smg7 reduces Ferrapoptosis and promotes neurological recovery after TBI in mice. Our work systematically delineates a previously unrecognized form of cell death and its pathogenetic mechanisms, providing experimental evidence to refine cell death classification, and suggesting a conceptual strategy for treating complex diseases such as TBI by targeting shared regulatory nodes. HIGHLIGHTS Defined and validated a previously unrecognized cell death modality, termed “Ferrapoptosis”. Elucidated the ordered pattern of Ferrapoptosis across key stages of TBI pathogenesis. Revealed mitochondrial oxidative stress as the pivotal hub integrating ferroptosis and apoptosis. Identify Smg7 as a core driver of Ferrapoptosis and a promising neuroprotective target.

Salinity stress threatens rice (Oryza sativa L.) production across 1.056 million hectares of Bangladesh's coastal regions, with intensification projected due to climate change and sea-level rise. Marker-assisted breeding (MAB) has emerged as a transformative approach for developing salt-tolerant varieties, offering precision and efficiency over conventional methods. This review examines marker-assisted selection (MAS) and marker-assisted backcrossing (MABC) applications for salt tolerance improvement in Bangladesh's rice breeding programs. We analyze the genetic architecture of salt tolerance, particularly the major Saltol QTL harboring OsHKT1;5 on chromosome 1, and discuss molecular marker systems including SSRs, SNPs, and InDels utilized by the Bangladesh Rice Research Institute (BRRI). The review documents successful introgression of salt tolerance into elite varieties, resulting in seventeen released varieties including BRRI dhan47, BRRI dhan73, and BRRI dhan78, with yields of 4.5-8.3 t ha⁻¹ under saline conditions (8-14 dS m⁻¹). We evaluate physiological mechanisms of Na⁺ exclusion and ionic homeostasis, analyze breeding achievements using foreground and background selection strategies, and discuss future integration of genomic selection, GWAS, and CRISPR/Cas9 gene editing. Despite substantial progress, continued integration of molecular breeding with phenomics and participatory approaches remains essential for developing climate-resilient varieties addressing Bangladesh's intensifying salinity challenge.

Summary The actin-binding protein synaptopodin (Synpo) regulates the cytoskeleton and intracellular Ca 2+ and is important for long-term potentiation (LTP) and learning. The inconsistent onset age for LTP in mice makes their Synpo knockout (KO) a suboptimal developmental model. Hence, we generated Synpo KO rats using CRISPR-Cas9. Synpo KO rats are viable with reduced body weight and bone length after postnatal days (P)35-P45. Their basal kidney function is normal. 3D reconstruction from electron microscopy reveals the absence of the Synpo-dependent dendritic spine apparatus and cisternal organelles in the axon initial segment (AIS), which may contribute to reduced LTP in the KO rat. Inhibitory synapses in the wild-type AIS appear preferentially clustered near cisternal organelles—a pattern disrupted in the KO, where synapses appear more uniformly distributed. The consistent developmental profile of LTP in the rat makes this KO a robust model to assess Synpo function in development, synaptic plasticity, and behavior. Graphical Abstract