Zebrafish (Danio rerio) has become a pivotal vertebrate model in biomedical research, renowned for its genetic similarity to humans, optical transparency, rapid embryonic development, and amenability to experimental manipulation. In recent years, the derivation of cell lines from zebrafish embryos has unlocked new possibilities for in vitro studies across developmental biology, toxicology, disease modeling, and genetic engineering. These embryo-derived cultures offer scalable, reproducible, and ethically favorable alternatives to in vivo approaches, enabling high-throughput screening and mechanistic exploration under defined conditions. This review provides a comprehensive overview of protocols for establishing and maintaining zebrafish embryonic cell lines, emphasizing culture conditions, pluripotency features, transfection strategies, and recent innovations such as genotype-defined mutant lines generated via CRISPR/Cas9 and feeder-free systems. We also highlight emerging applications in oncology, regenerative medicine, and functional genomics, positioning zebrafish cell lines as versatile platforms bridging animal models and next-generation in vitro systems. Their ongoing optimization holds promise for improved reproducibility, reduced animal use, and expanded translational impact in biomedical research.

Summary Replication stress is a key driver of DNA damage and genome instability. Replication stress-induced fork remodelling generates a new DNA end that is vulnerable to the action of nucleases, and which is protected by a range of factors including the canonical tumour suppressors BRCA1 and BRCA2. Here we report that replication stress drives elevated production of cytokines and chemokines in the absence of DNA damage. The DNA sensor IFI16 binds nascent DNA at stalled replication forks and signals via the DNA sensing adaptor STING, to induce the activation of NF-κB and the production of pro-inflammatory cytokines in response to replication stress. IFI16 also acts directly at stalled replication forks to protect nascent DNA from degradation by the nucleases MRE11 and DNA2. Furthermore, IFI16 is required for the interferon-mediated rescue of fork protection in BRCA-deficient cells, highlighting the critical role of IFI16 in the cross-talk between innate immunity and fork protection during replication stress. Highlights Replication stress induces an early innate immune response, which is dependent on the DNA sensing factors IFI16 and STING, but not cGAS IFI16 binds directly to nascent DNA at stalled replication forks IFI16 prevents nucleolytic degradation of reversed forks IFI16 is required for interferon-mediated fork protection in BRCA-deficient cells

Cell transfer experiments complement the rigorous investigation of antiviral and antitumor functions of natural killer (NK) cells. Success in these endeavors is enhanced by expansion of small numbers of input NK cells driven by viral antigens or homeostatic proliferation in immunodeficient hosts. In contrast, analysis of other NK-cell functions, including immunoregulation, are non-proliferative and require an intact immune system in recipient mice. We reveal poor persistence of conventional congenic (CD45.1) BoyJ NK cells following adoptive transfer in comparison to CRISPR-generated CD45.1+ (JAXBoy) NK cells. Reciprocal transfers between C57BL/6 and JAXBoy mice substantially improve seeding and maintenance of donor NK cells. Using this system, we confirm that CXCR3 re-positions NK cells in the white pulp of the spleen after infection, which is vital for immunoregulation. Moreover, we discovered that the transcription factor ASCL2 is required for recruitment of NK cells into the spleen and white pulp. These results provide improved tools and novel insights into NK cell biology. Key points JAXBoy are superior to conventional BoyJ mice for NK cell persistence after transfer. CXCR3 repositions donor NK cells in T/B-zones of the spleen after infection. The transcription factor ASCL2 is required for NK-cell recruitment to spleen white pulp.

Guide RNA (gRNA) arrays can enable targeting multiple genomic loci simultaneously using CRISPR-Cas9. In this study, we present a streamlined and efficient method to rapidly construct gRNA arrays with up to 10 gRNA units in a single day. We demonstrate that gRNA arrays maintain robust functional activity across all positions, and can incorporate libraries of gRNAs, combining scalability and multiplexing. Our approach will streamline combinatorial perturbation research by enabling the economical and rapid construction, testing, and iteration of gRNA arrays.

Several members of the epidermal growth factor (EGF) family have been implicated in the biology of schizophrenia (Ketharanathan et al., 2024). The EGF-related ligand, Betacellulin (BTC), plays an important role in the proliferation and differentiation of neural stem cells and our group found markedly reduced BTC levels in patients with schizophrenia. Nevertheless, the interplay of affected BTC and its participation in neural specification and neurodevelopment remains elusive. We generated Knockout (KO) - BTC clones from an existing hiPSC line through CRISPR/Cas9-mediated modification. Furthermore, we validated BTC-KO through genotyping/sequencing, FACS and Western Blot. Finally, we demonstrated trilineage differentiation potential in vitro .

The CRISPR/Cas9 system is a powerful gene-editing tool. Its specificity and stability rely on complex allosteric regulation. Understanding these allosteric regulations is essential for developing high-fidelity Cas9 variants with reduced off-target effects. Here, we introduce a novel structure-based machine learning (ML) approach to systematically identify long-range allosteric networks in Cas9. Our ML model was trained using all available Cas9 structures, ensuring a comprehensive representation of Cas9 structural landscape. We then applied this model to Streptococcus pyogenes Cas9 (SpCas9) to demonstrate the feature selection process. Using the Cα-Cα inter-residue distances, we mapped key allosteric networks and refined them through a two-stage SHAP feature selection (FS) strategy, reducing a vast feature space to 28 critical Lysine-Arginine (Lys-Arg) residue pairs that mediate SpCas9 interdomain communication, stability, and specificity. These Lys-Arg pairs initially shared a 46.5Å inter-residue distance, but molecular dynamics simulations revealed distinct stabilization behaviors, indicating a hierarchical allosteric network. Further mutational analysis of R78A-K855A (M1) and R765A-K1246A (M2) identified an electrostatic valley, a stabilizing network where positively charged residues interact with negatively charged DNA to maintain SpCas9 structural integrity. Disrupting this valley through direct (M2) or allosteric (M1) mutations destabilized SpCas9 DNA-bound conformation, leading to distinct pathways for improving SpCas9 specificity. This study provides a new framework for understanding allostery in Cas9, integrating ML-driven structural analysis with MD simulations. By identifying key allosteric residues and introducing the electrostatic valley as a central concept, we offer a rational strategy for engineering high-fidelity Cas9 variants. Beyond Cas9, our approach can be applied to uncover allosteric hotspots in other enzyme regulation and rational protein design.

Identification of new therapeutic targets in hepatocellular carcinoma (HCC) remains critical. Chromatin regulating complexes are frequently mutated or aberrantly expressed in HCC, suggesting dysregulation of chromatin environments is a key feature driving liver cancer. To investigate whether the altered chromatin state in HCC cells could be targeted, we designed and utilized an epigenome-focused CRISPR library that targets genes involved in chromatin regulation. This focused approach allowed us to test multiple HCC cell lines in both 2D and 3D growth conditions, which revealed striking differences in the essentiality of genes involved in ubiquitination and multiple chromatin regulators vital for HCC cell survival in 2D but whose loss promoted growth in 3D. We found the core subunits of the menin-MLL1 complex among the strongest essential genes for HCC survival in all screens and thoroughly characterized the mechanism through which the menin-MLL1 complex promotes HCC cell growth. Inhibition of the menin-MLL1 interaction led to global changes in occupancy of the complex with concomitant decreases in H3K4me3 and expression of genes involved in PI3K/AKT/mTOR signaling pathway. Menin inhibition affected chromatin accessibility in HCC cells, revealing that increased chromatin accessibility at sites not bound by menin-MLL1 was associated with the recruitment of the pioneer transcription factor complex NF-Y. A CRISPR/Cas9 screen of chromatin regulators in the presence of menin inhibitor SNDX-5613 revealed a significantly increased cell death when combined with NFYB knockout. Together these data show that menin-MLL1 is necessary for HCC cell survival and cooperates with NF-Y to regulate oncogenic gene transcription.

DNA methyltransferase 1 inhibitor (DNMT1i) therapy is a promising option for increasing immune response as part of combination cancer therapy. High–grade serous ovarian carcinoma (HGSOC) is a highly aggressive cancer with poor survival outcomes, where DNMT1i therapy is being increasingly explored. HGSOC with epigenetically silenced BRCA1 has been shown to respond to PARP inhibitor (PARPi) treatment – a core targeted therapy for HGSOC. However, loss of silencing of even a single BRCA1 allele causes PARPi and platinum chemotherapy resistance. We tested whether BRCA1 silencing was robust to DNMT1i therapy, or would be reversed, thus driving PARPi resistance. We previously generated two homozygously silenced BRCA1 HGSOC cell lines: WEHI–CS62 and an OVCAR8 derivative. DNMT1i treatment caused sustained BRCA1 promoter methylation loss, gene re–expression and PARPi resistance in both of these silenced BRCA1 lines, but not in mutated BRCA1/2 or RAD51C lines. Methylation arrays confirmed transient global CpG methylation losses following DNMT1i. CRISPR deletion of the re–expressed BRCA1 copy in WEHI-CS62 restored silencing and PARPi sensitivity. Furthermore, DNMT1i treatment of a silenced BRCA1 PDX caused heterogeneous BRCA1 promoter methylation loss. In summary, DNMT1 inhibitors caused sustained reduction of BRCA1 promoter methylation in HGSOC cells. This resulted in BRCA1 re-expression and PARP inhibitor resistance, presenting a significant risk for up to 17% of HGSOC patients with BRCA1 gene silencing who could benefit from PARP inhibitor therapy. We conclude that DNA demethylation therapy should be avoided for HGSOC patients with epigenetically silenced BRCA1.

ABSTRACT Spatial RNA organization plays a pivotal role in diverse cellular processes and diseases, but the functional implications of spatial RNA localization remain underexplored. We present CRISPR-mediated transcriptome o rganization (CRISPR-TO) that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of RNA localization in live cells. CRISPR-TO enables targeted localization of RNAs to diverse subcellular compartments, including p-bodies, stress granules, telomeres, and nuclear stress bodies, across cell types. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips and co-transport with ribosomes, with β -actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. Furthermore, CRISPR-TO-enabled parallel screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by current sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.

Abstract Background: Dissecting the functional impact of genetic mutations is essential to advancing our understanding of genotype-phenotype relationships and identifying new therapeutic targets. Despite the progress in sequencing and CRISPR technologies, proteome-wide mutation effect prediction remains challenging. Here, we introduce ProteoCast, a scalable and interpretable computational method for proteome-wide classification of genetic variants and functional protein site identification. It relies solely on evolutionary information, leveraging protein sequence data across organisms. Results: Using ProteoCast, we generated mutational landscapes for 22,169 Drosophila melanogaster protein isoforms, categorising over 293 million amino acid substitutions as functionally neutral, uncertain, or impactful. We validated our predictions with over 380 thousand natural polymorphisms observed in the Drosophila Genetic Reference Panel (DGRP) and Drosophila Evolution over Space and Time (DEST) datasets and with FlyBase's developmentally lethal mutations. About 86% of known lethal mutations were classified as impactful or uncertain, versus only 13% and 18% of DGRP and DEST mutations. Moreover, we performed ProteoCastguided genome editing experiments, providing a proof-of-concept of the validity of this strategy. Beyond variant effect prediction, ProteoCast detected evolutionary conservation signals in about one-third of 40.5K annotated post-translational modification sites and 83% of ~90 known short linear motifs. These results support its usefulness for uncovering interaction and regulatory sites in unstructured protein regions. Conclusions: Our results demonstrate ProteoCast applicability for model organisms, contributing to basic genetic research and translational studies. This work provides a publicly available dataset, userfriendly interactive web services, and a locally deployable pipeline tool for further research into gene function and mutation effects in any organism.

Enzymatic browning and cold-induced sweetening (CIS) affect the post-harvest quality of potato tubers. Browning is caused by Polyphenol Oxidase 2 (PPO2), which is activated by mechanical damage during harvest and storage. CIS occurs when vacuolar invertase converts sucrose into reducing sugars, which react with amino acids during frying, forming brown pigments and acrylamide. While cold storage prevents sprouting and disease, it also increases vacuolar invertase expression, leading to quality loss. Using CRISPR/Cas9, we developed gene-edited potato lines with improved resistance to browning and CIS. Line 6A (cv. Atlantic) and E03-3 (cv. Spunta) exhibited complete vacuolar invertase (InvVac) knockout, maintaining chip quality for at least 60 days at 4°C. Line 6A, renamed PIRU INTA, was tested in field trials and preserved frying quality for up to 90 days under cold storage. PIRU INTA is currently undergoing registration as a new variety. Additionally, lines E04-5B and E03-3 (cv. Spunta) showed partial PPO2 gene edits, reducing enzymatic browning by 80% and 40%, respectively. This study demonstrates the potential of CRISPR/Cas9 to develop non-transgenic, gene-edited potatoes with enhanced storage quality, benefiting both growers and the food industry.

Hepatic stellate cells (HSCs) are critical for normal liver development and regeneration. Podocalyxin-like (podxl) is highly expressed in zebrafish HSCs, but its role in liver development is not known. Here we report that podxl knockdown using CRISPR/Cas9 (“CRISPants”) significantly decreased HSC number in zebrafish larvae at different time points and in two independent HSC reporter lines, supporting a role for podxl in HSC development. We generated five podxl mutants, including two mutants lacking the predicted podxl promoter region, and found that none of the mutants recapitulated the knockdown phenotype. Podxl CRISPR/Cas9 injection in mutants lacking the podxl guide RNA cut site did not affect HSC number, supporting the hypothesis that the CRISPant phenotype was specific, requiring intact podxl. Podxl mRNA levels in three podxl mutants were similar to those of wildtype controls. RNA sequencing of podxl mutants and controls showed no significant change in transcript levels of genes with sequence similarity to podxl , but it revealed upregulation of a network of extracellular matrix genes in podxl mutants. These results support a role for podxl in zebrafish liver development and suggest that upregulation of a group of functionally related genes represents the main mechanism of compensation for podxl genomic loss.

ABSTRACT The Gram-negative anaerobe Fusobacterium nucleatum is an oral oncobacterium that promotes colorectal cancer (CRC) development with the amyloid-forming cell surface adhesin FadA integral to CRC tumorigenesis. We describe here molecular genetic studies uncovering a novel mode of metabolic regulation of FadA-mediated tumor formation by a highly conserved respiratory enzyme known as the Rnf complex. First, we show that genetic disruption of Rnf, via rnfC deletion, significantly reduces the level of fadA transcript, accompanied by a near-complete abolishment of the precursor form of FadA (pFadA), reduced assembly of FadA at the mature cell pole, and severe defects in the osmotic stress-induced formation of FadA amyloids. We show further that the Rnf complex regulates three response regulators (CarR, ArlR, and S1), which modulate the expression of pFadA, without affecting fadA transcript. Consistent with our hypothesis that these response regulators control factors that process FadA, deletion of rnfC , carR , arlR , or s1 each impairs expression of the signal peptidase gene lepB , and FadA production is nearly abolished by CRISPR-induced depletion of lepB . Importantly, while rnfC deletion does not affect the ability of the mutant cells to adhere to CRC cells, rnfC deficiency significantly diminishes the fusobacterial invasion of CRC cells and formation of spheroid tumors in vitro . Evidently, the Rnf complex modulates the expression of the FadA adhesin and tumorigenesis through a gene regulatory network consisting of multiple response regulators, each controlling a signal peptidase that is critical for the post-translational processing of FadA and surface assembly of FadA amyloids. IMPORTANCE The R hodobacter n itrogen-fixation (Rnf) complex of Fusobacterium nucleatum plays an important role in the pathophysiology of this oral pathobiont, since genetic disruption of this conserved respiratory enzyme negatively impacts a wide range of metabolic pathways, as well as bacterial virulence in mice. Nonetheless, how Rnf deficiency weakens the virulence potential of F. nucleatum is not well understood. Here, we show that genetic disruption of the Rnf complex reduces surface assembly of adhesin FadA and FadA-mediated amyloid formation, via regulation of signal peptidase LepB by multiple response regulators. As FadA is critical in the carcinogenesis of colorectal cancer (CRC), the ability to invade CRC cells and promote spheroid tumor growth is strongly diminished in an Rnf-deficient mutant. Thus, this work uncovers a molecular linkage between the Rnf complex and LepB-regulated processing of FadA – likely via metabolic signaling – that maintains the virulence potential of this oncobacterium in various cellular niches.

Abstract The CRISPR/Cas9 system facilitates precise genome editing in various organisms. In this study, a single-vector CRISPR/Cas9 system was developed for Saccharomyces cerevisiae , employing a type II Cas9 enzyme from Streptococcus pyogenes and a single-guide RNA cassette targeting CAN1.Y locus on chromosome V. This system is broadly applicable across yeast strains, as it utilizes G418 selection, eliminating the need for auxotrophic markers. The efficiency of the CRISPR/Cas9 system was demonstrated, with editing efficiencies ranging from 70–100%. This system was utilized to integrate a cassette encoding secretory pectate lyase (PL) from Bacillus subtilis 168 into the yeast genome. The engineered S. cerevisiae strain secreted active PL, which exhibited pectin-degrading activity characterized by significant reductions in residual pectin and increased production of reducing sugars. Since pectin constitutes a major component of coffee mucilage, the secreted PL was applied to coffee beans for mucilage removal. The treated beans presented noticeably reduced residual mucilage, a purer green color, and decreased viscosity. These findings suggest the potential of the engineered S. cerevisiae strain for applications in coffee processing, particularly in efficient mucilage removal.

Plasmids are major drivers of microbial evolution, enabling horizontal gene transfer (HGT) and facilitating adaptation through the dissemination of relevant functional genes and traits. However, little is known about plasmid diversity and function in extremophiles. ‘ Fervidacidithiobacillus caldus’, a meso-thermo-acidophilic sulfur oxidizer, is a key player in sulfur cycling in natural and industrially engineered acidic environments. Here, we present a comprehensive analysis of the plasmidome, and associated anti-mobile genetic element (anti-MGE) defense systems (defensome), across genomes of this species and metagenomes from diverse natural and industrial settings harboring ‘ F. caldus’ . We identified >30 distinct plasmids, representing five consistent replication-mobilization families. Plasmids ranged in size between 2.5–65 kb, with gene content and plasmid modularity scaling with element size and copy numbers inversely correlating with size. Plasmids carried variable numbers of hypothetical proteins and transposases, with annotated cargo genes reflecting functional differentiation by habitat. Defensome profiling revealed over 50 anti-MGE systems in sequenced ‘ F. caldus’ isolates, including diverse restriction-modification systems, CRISPR-Cas types IV-A and V-F, and widespread abortive infection and composite defense systems such as Wadjet, Gabija, and Zorya. In environmental populations, an inverse relationship was observed between defensome complexity and plasmidome abundance and diversity, underscoring a pivotal role of the host defensome in modulating persistence, compatibility, and overall plasmid diversity across ‘ F. caldus’ populations. Yet, other plasmids appeared decoupled from both host abundance and defensome complexity, suggesting potential host shifts, environmental persistence, or differential replication under suboptimal growth conditions for the host. Altogether, these findings reveal a modular, adaptive plasmidome shaped by selective pressures and host–plasmid–defensome interactions and positions plasmids as key contributors to adaptation, gene flow, and functional innovation in this extreme acidophile. Importance Plasmids are key vehicles of gene exchange and adaptation in bacteria, yet their roles in extremophilic systems remain poorly understood. This study provides the first integrated view of the plasmidome and defense systems in ‘ Fervidacidithiobacillus caldus’ , a sulfur-oxidizing acidophile relevant to both natural biogeochemical cycling and industrial bioleaching. We uncover a rich plasmid diversity structured into modular families with variable cargo and backbone features and reveal their coexistence with complex anti-MGE defense repertoires. By combining genomic and metagenomic approaches, we expose principles of plasmid compatibility, persistence, and habitat-specific adaptation. These insights expand current knowledge of mobile genetic elements in extreme environments and provide a foundation for plasmid-based vector design and synthetic biology in acidophiles, with direct implications for biomining and environmental remediation in extreme environments.

Abstract Background Long dismissed as mere genomic parasites, transposable elements (TEs) are now recognized as major drivers of genome evolution. TEs serve as a source of cell-type specific cis -regulatory elements, influencing gene expression and observable phenotypes. However, the precise TE regulatory roles in different contexts remain largely unexplored and the impact of TEs on transcriptional regulatory networks and contribution to disease risk is likely deeply underestimated. Results Using a multimapper-aware strategy, we systematically characterised the epigenetic profile of TEs in the brain. This analysis revealed that MER57E3, a primate-specific TE subfamily, exhibits strong enrichment for active, and absence of repressive, histone modifications across six brain cell types. MER57E3 copies are predominantly located near zinc finger genes and enriched for homeodomain motifs recognized by brain-specific transcription factors, including GBX1 and BSX. Upon CRISPR interference (CRISPRi) targeting specific MER57E3 copies, RNA-seq analysis demonstrated downregulation of the key neurogenesis-related genes PAX6 and NEUROG2 . Conclusions Our data indicate that members of the MER57E3 TE subfamily regulate the expression of critical neurogenesis genes during neural progenitor cell (NPC) development. Moreover, this study emphasises the importance of characterising TEs, offering new insights into how their epigenetic dysregulation may contribute to pathogenesis of neurodevelopmental disorders.

Background & Aims Somatic and germline CIDEB mutations are associated with protection from chronic liver diseases. The mechanistic basis and whether CIDEB suppression would be an effective therapy against fatty liver disease remain unclear. Methods 21 CIDEB somatic mutations were introduced into cells to assess functionality. In vivo screening was used to trace Cideb mutant clones in mice fed normal chow, western (WD), and choline-deficient, L-amino acid-defined, high-fat (CDA-HFD) diets. Constitutive and conditional Cideb knockout mice were generated to study Cideb in liver disease. Isotope tracing was used to evaluate fatty acid oxidation and de novo lipogenesis. Transcriptomics, lipidomics, and metabolic analyses were utilized to explore molecular mechanisms. Double knockout models ( Cideb/Atgl and Cideb/Ppara ) tested mechanisms underlying Cideb loss. Results Most CIDEB mutations showed that they impair function, and lineage-tracing showed that loss-of-function clones were positively selected with some, but not all fatty liver inducing diets. Cideb KO mice were protected from WD, CDA-HFD, and alcohol diets, but had the greatest impact on CDA-HFD induced liver disease. Hepatocyte-specific Cideb deletion could ameliorate disease after MASLD establishment, modeling the impact of therapeutic siRNAs. Cideb loss protected livers via increased β-oxidation, specifically through ATGL and PPARa activation. Conclusions Cideb deletion is more protective in some types of fatty liver disease. β-oxidation is an important component of the Cideb protective mechanism. CIDEB inhibition represents a promising approach, and somatic mutations in CIDEB might predict the patient populations that might benefit the most.

Craniofacial development and neural crest specification are evolutionarily conserved processes, yet subtle modifications to their gene regulatory networks drive species-specific craniofacial diversity. Transposable elements (TEs) are increasingly recognized as contributors to genome evolution, but their role in shaping neural crest regulatory programs remains underexplored. Here, we investigate the domestication of human-specific TEs as transcriptional enhancers during cranial neural crest cell (CNCC) specification, a process critical for vertebrate head development. Using human iPSC-derived CNCCs, we identified ∼250 human-specific TEs acting as active enhancers. These TEs were predominantly LTR5Hs and, to a lesser extent, SVA-E/Fs. We demonstrate that these elements have been co-opted through the acquisition of the conserved CNCC coordinator motif, and are bound by the CNCC signature factor TWIST1, and that their co-option appears to be largely exclusive to CNCCs. To assess their functional relevance, we used CRISPR-interference to repress ∼75% of all the LTR5Hs and SVAs active in CNCCs, which led to widespread transcriptional changes in genes associated with neural crest migration, a process essential for CNCCs to populate the embryo and form craniofacial structures. Using a cell migration assay, we showed that CNCC migration was significantly impaired by CRISPR-mediated TE repression. Finally, we demonstrate that genes near human-specific TEs are more highly expressed in human CNCCs relative to chimpanzee, and TE repression returns their expression to chimpanzee levels. These findings reveal how human-specific TEs have been co-opted to fine-tune CNCC regulatory networks, potentially contributing to the evolution of lineage-specific craniofacial traits.

Tick-borne orthoflaviviruses (TBOVs) are a growing global health concern. Several representatives of this viral family cause fatal disease in humans with increasing case numbers throughout the last decades. The innate immune response, especially interferon (IFN)-dependent signaling, is an essential part of the human defense system that counteracts infection with TBOVs and other viruses. Even though they activate the same signaling cascade, IFNs belonging to the type I and III families trigger differing gene expression patterns. Which genes the two IFN families induce to restrict infection with TBOVs remains poorly characterized. Here we show that type I and III IFNs are both capable of restricting TBOV infection of human cell lines in a cell type-specific manner. Infection of C57BL/6J mice with knockouts for either IFN type I or III receptors further underscored the critical role of IFN signaling in controlling TBOV replication in vivo . To assess the contribution of single genes to controlling TBOV infection in human cells, we used a CRISPR/Cas9-KO-based screening approach. This strategy identified IFI6 as a central player for IFN type I- and III-driven responses against TBOVs. We further defined IFI6 as an ER-resident protein that restricts TBOV replication at a post-entry step. Our work thus opens new perspectives for targeting weak points in the life cycle of TBOVs and other orthoflaviviruses, potentially paving the way for the development of new antiviral therapeutics. One Sentence Summary Type I and III interferons are crucial for protection against tick-borne orthoflavivirus infection in vitro and in vivo , both relying on IFI6 as a main antiviral effector.

ABSTRACT Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platform’s performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of SARS-CoV-2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research. GRAPHICAL ABSTRACT

Tumors require metabolic adaptations to support their rapid growth, but how they influence lipid metabolism in distant tissues remains poorly understood. Here, we uncover a novel mechanism by which gut tumors in adult flies reprogram lipid metabolism in distal hepatocyte-like cells, known as oenocytes, to promote tracheal development and tumor growth. We show that tumors secrete a PDGF/VEGF-like factor, Pvf1, that activates the TORC1-Hnf4 signaling pathway in oenocytes. This activation enhances the production of specific lipids, including very long-chain fatty acids and wax esters, that are required for tracheal growth surrounding the gut tumor. Importantly, reducing expression in oenocytes of either the transcription factor Hnf4 , or the elongase mElo that generates very long chain fatty acid suppresses tumor growth, tracheogenesis, and associated organ wasting/cachexia-like phenotypes, while extending lifespan. We further demonstrate that this regulatory pathway is conserved in mammals, as VEGF-A stimulates lipid metabolism gene expression in human hepatocytes, and lung tumor-bearing mice show increased hepatic expression of Hnf4 and the lipid elongation gene Elovl7 . Our findings reveal a previously unrecognized tumor-host interaction where tumors non-autonomously reprogram distal lipid metabolism to support their growth. This study not only identifies a novel non-autonomous role of the TORC1-Hnf4 axis in lipid-mediated tumor progression but also highlights potential targets for therapeutic intervention in cancer-associated metabolic disorders.

ABSTRACT Capillary malformations (CM) are slow-flow vascular abnormalities present at birth and predominantly manifest as cutaneous lesions. In the rare neurocutaneous disorder known as Sturge Weber Syndrome (SWS), individuals exhibit CM not only on the skin but also within the leptomeninges of the brain and the choroid of the eye. >90% of CM are caused by a somatic R183Q mutation in GNAQ, the gene encoding Gαq – a heterotrimeric G-protein subunit. The somatic GNAQ mutation is notably enriched in endothelial cells (ECs) isolated from CM-affected regions. Here we show blood vessels in cutaneous and leptomeningeal SWS lesions exhibit extravascular fibrin indicating a compromised endothelial barrier. Longitudinal MRI of the brain in one SWS patient further suggests vascular permeability. To explore this pathological phenotype, we employed the trans-endothelial electrical resistance (TEER) assay to measure permeability of the EC-EC barrier in vitro . Human EC CRISPR edited to create a GNAQ R183Q allele (EC-R183Q) exhibited a reduced barrier compared to mock edited EC (EC-WT). We sought to identify signaling molecules needed for EC barrier formation. Knockdown of angiopoietin-2 (ANGPT2), known to be significantly increased in EC-R183Q and in CM, partially yet significantly restored the barrier, while an anti-ANGPT2 function blocking antibody did not. We next tested the MEK1,2 inhibitor (Trametinib) because MAPK signaling is increased by GNAQ mutation. MEK1,2 inhibitors partially restored the EC barrier, implicating involvement of MAPK/ERK signaling. The combination of ANGPT2 knockdown and Trametinib significantly restored the EC barrier to near EC-WT levels. The additive impacts of ANGPT knockdown and MEK1,2 inhibition indicate the two operate in separate pathways. In summary, we discovered that GNAQ p.R183Q ECs exhibit compromised endothelial barrier formation, reflecting the compromised EC barrier in CM lesions, and that ANGPT2 knockdown combined with Trametinib effectively restores the EC-EC barrier. NONSTANDARD ABBREVIATIONS AND ACRONYMS NOVELTY AND SIGNIFICANCE What is known? The mutant Gαq-R183Q in endothelial cells activates phospholipase β3, contributing to increased angiopoietin-2, a pro-angiogenic, proinflammatory molecule that contributes to vascular permeability. Endothelial Gαq-R183Q is sufficient to drive formation of enlarged blood vessels akin to what is observed in CM. ANGPT2 shRNA knockdown prevented the enlarged vessel phenotype in a xenograft model. An EC-specific GNAQ p.R183Q mouse model showed permeability in brain vessels, detected by perfusion of Evans Blue dye, indicating reduced vascular integrity. What New Information Does This Article Contribute? Reduced vascular integrity in CM is confirmed by Martius Scarlet Blue staining and longitudinal MRI imaging of SWS brain. GNAQ p.R183Q EC form a weaker endothelial barrier in vitro compared to control ECs. The weakened endothelial barrier in the mutant ECscan be rescued by Gαq inhibitor, YM254890, confirming the compromised barrier is a consequence of the mutant Gαq. Titration experiments modeling the mosaic nature of the GNAQ p.R183Q in CMshow that 5- 10% GNAQ p.R183Q EC in the monolayer is sufficient to reduce endothelial barrier formation. Knockdown of ANGPT2 or MEK1,2 inhibition partially restored the endothelial barrier in GNAQ p.R183Q EC. Combining knockdown of ANGPT2 and addition of a MEK inhibitor, Trametinib, restored the endothelial barrier to near what is seen in wild type ECs. What is the translational message? Sturge Weber Syndrome (SWS) is a neurocutaneous disorder that involves atypical blood vessel overgrowth in the skin, brain and eye. It is associated with facial CM (aka port wine birthmark), leptomeningeal CM in the brain visible with MRI, and glaucoma. Theneurological sequalae involve seizures, cerebral atrophies and calcification, and intellectual disorders. Currently there are no molecularly targeted therapies for non-syndromic CM or SWS. Our study shows the involvement of MAPK pathway and the proinflammatory molecule ANGPT2 in endothelial permeability and suggests a path to target GNAQ p.R183Q driven CM.

Genetic risk for psychiatric disorders lies largely within non-coding regions, where the lack of detailed knowledge of gene regulation and chromatin structure has hampered understanding of disease mechanisms. We analyzed chromatin accessibility and 3D genome architecture in brains from 53 ASD and neurotypical individuals, including patients with (dup) 15q11-13. We observed reduced CTCF binding, which had dual effects: a) decreased chromatin accessibility at distal enhancers and downregulation of synaptic and neuronal target genes, and b) weakened TAD boundaries linked to DNA hypermethylation, impacting a distinct set of genes. These changes were associated with brain mQTLs, caQTLs, and rare variants increasing ASD risk, a subset of which we validated by CRISPR editing, supporting a causal relationship. Our analyses suggest that genetic variants contribute to risk in part through a combination of epigenetic changes, including disruption of distal enhancer accessibility and 3D genome organization in both idiopathic and a syndromic form of ASD.

Abstract Restrictive cardiomyopathy (RCM) is a rare, fatal disorder that rapidly progresses in children. TNNI3 mutations represent the most common genetic cause. Although cTnI mutations are known to increase myofilament calcium sensitivity and impair diastolic function, this mechanism alone does not fully account for disease pathogenesis. Recent studies have revealed that the immune system plays an important role in cardiovascular diseases, however, its involvement in RCM remains unclear. Here, we generated a classic cTnIR193H mouse model using CRISPR/Cas9. Cardiac RNA-seq analysis indicated marked activation of innate immune pathways. moreover, biotin-mediated proximity labeling combined with quantitative mass spectrometry identified differential interactors of the cTnIR193H mutant, with Irgm1 emerging as the most significantly altered immune-related protein. Notably, the cTnIR193H mutation enhances binding to Irgm1 without affecting its expression, thereby indirectly inhibiting its normal function. This aberrant interaction activates the cGAS-STING pathway and elicits a type I interferon response in the hearts of RCM mice. Furthermore, treatment with the STING inhibitor C176 partially restored diastolic function and significantly alleviated cardiac fibrosis. Taken together, this study reveals for the first time that immune mechanisms play a crucial role in RCM pathogenesis and provides a potential therapeutic target for RCM treatment from an immunological perspective.

Tight control of mesenchymal cell migration is important for embryonic development and its deregulation causes disease. It is driven by lamellipodia protrusion, the leading edge of the migrating cell. This is controlled by Rac-Scar/WAVE-Arp2/3 complexes driving actin filament nucleation coupled to Ena/VASP proteins mediating actin filament elongation. These activities are coordinated by leading-edge proteins, such as Lamellipodin and NHSL1. Here, we discovered KIAA1522/NHSL3 as an additional regulator of these essential actin effectors. We reveal that NHSL3 promotes cell migration. NHSL3 co-localises at the edge of lamellipodia with Ena/VASP proteins and the Scar/WAVE complex. We show that it binds to Ena/VASP proteins and the Scar/WAVE complex and functions to inhibit Scar/WAVE-Arp2/3 activity in cells. NHSL3 interacts with the Scar/WAVE complex subunit Abi and, in contrast to other known Scar/WAVE complex binders, additionally to the CYFIP1/2 subunit through 3 short linear motifs. Thus, control of actin filament nucleation and elongation at the leading edge of mesenchymal cells is more complex than anticipated. Our study provides insights into the intricate regulation of lamellipodial actin networks highly relevant for understanding control of mesenchymal cell migration during development and diseases.