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.

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.