We present Alpha-BET, a structure-guided strategy leveraging AlphaFold to identify optimal ALFA-tag insertion sites for minimally disruptive labeling of viral glycoproteins with high-affinity nanobodies. Applied to HIV-1 Env, SARS-CoV-2 S, and NiV G, Alpha-BET preserves structural integrity and function. For HIV-1 Env, we demonstrate super-resolution DNA-PAINT MINFLUX 3D imaging enabled by tag insertion, showcasing its power for visualizing native trimers in single virions and potential for broader applications in virus research.

Abstract Genome editing by CRISPR-Cas9 is promising for genetic disease and cancer gene therapy. However, safety concerns are still present, particularly the ON-target genotoxicity for protocols using nucleases. Quality control of edited cells before and after graft is mandatory, especially to assay megabase-scale genomic rearrangements induced at the targeted locus. These unintended events are fortunately rare but potentially deleterious. Classical PCR-based bulk approaches do not detect them or underestimate their frequency. Single-cell approaches are promising but RNA sequencing only estimates copy number variation of the genome. Here, we propose single-cell DNA sequencing to accurately evaluate and monitor CRISPR-mediated genotoxicity in primary cells (human fibroblasts and hematopoietic stem/progenitor cells). We designed a homemade panel using single nucleotide polymorphisms to detect, map and characterize induced-losses of heterozygosity (terminal, interstitial, copy-loss and copy-neutral). This innovative approach revealed intense genotoxicity linked to the double strand break. The risk was associated with DNA repair modulators in particular DNA-PKcs inhibitor AZD7648. Importantly, the CDK4/6 inhibitor palbociclib prevented these rearrangements in hematopoietic stem/progenitor cells. This work strongly suggests that single-cell DNA sequencing should be routinely implemented in clinical applications before CRISPR-edited cell infusions.

Hemophilia B gene therapy treatments currently have not addressed the need for predictable, durable, active, and redosable factor IX (FIX). Unlike conventional gene therapy, engineered B Cell Medicines (BCMs) are durable, redosable, and titratable, and thus have the potential to address significant unmet needs in the Hemophilia B treatment paradigm. BE-101 is an autologous BCM comprised of expanded and differentiated B lymphocyte lineage cells genetically engineered ex vivo to secrete FIX-Padua. CRISPR/Cas9 mediated gene editing at the C-C chemokine receptor type 5 locus was used to facilitate transgene insertion of an AAV6-encoded DNA template via homology-directed repair. Transgene insertion did not alter B cell biology, viability, or differentiation into plasma cells. Appreciable levels of BE-101-derived FIX-Padua were detected within 1 day after IV administration in mouse and steady state was reached within 2 weeks and persisted for over 184 days. Redosing produced an increase in FIX-Padua production close to linear dose proportionality. Comprehensive genotoxicity analysis found no off-target issues of concern. No safety signals were observed in animal tolerability and GLP toxicology studies. In conclusion, BE-101 produces sustained levels of active FIX-Padua with the ability to engraft without host preconditioning and with the potential for redosing and titratability.

Simple Summary Diffuse midline gliomas (DMGs) are aggressive childhood brain tumours with no effective treatments. Over 80% of cases carry the histone H3K27M mutation, which alters chromatin structure and gene regulation and induce tumours growth. Immunotherapy, which uses the body’s immune system to fight cancer, relies on tumour- specific antigen molecules that immune cells can recognise. However, because DMGs have few mutations, finding suitable antigens for therapeutic purposes has been challenging. In this study, we investigated how the H3K27M mutation affects tumour antigen presentation in DMG. Using patient-derived DMG models, we found that H3K27M alters the landscape of antigens displayed on tumour cell surface, creating unique immune targets. We identified six immunogenic peptides, that triggered strong T cell responses. These antigens were absent when H3K27M was removed, confirming their link to the mutation. Our findings provide a blueprint for developing T cell-based immunotherapies for DMG, offering new hope for targeted treatments against this devastating disease. Background: Diffuse midline gliomas (DMGs) are among the most aggressive paediatric brain tumours, with the pathognomonic H3K27M mutation present in over 80% of cases. This mutation drives epigenetic dysregulation and transcriptional reprogramming, yet its impact on the tumour antigenic landscape remains poorly understood. Given the low mutational burden of DMG, an expanded search beyond neoantigens to include epigenetically dysregulated tumour-associated antigens (TAAs) is critical for advancing antigen-specific immunotherapies. Methods: To assess how H3K27M influences antigenic landscape of DMG, we performed a comprehensive immunopeptidomic analysis using patient-derived DMG cell line models (SU-DIPG13 and BT245) that harbour the H3K27M mutation and their CRISPR-edited H3K27M-knockout (KO) counterparts. High-resolution mass spectrometry and bioinformatics were employed to define H3K27M-driven changes in the immunopeptidome. Functional T cell assays using HLA-matched healthy donor PBMCs were conducted to evaluate the immunogenicity of H3K27M-associated peptides. Results: Our findings reveal that the H3K27M mutation reshapes the tumour antigenic landscape in a model-specific manner. While H3K27M knockout increased HLA-I expression in SU-DIPG13 but not BT245, immunopeptidomic profiling uncovered distinct shifts in the presentation of tumour-associated peptides, independent of direct effects on antigen processing machinery. Among these, we identified six immunogenic peptides, derived from SLITRK2, PRAME, XKR5, and CBX2, that elicited CD8⁺ T cell responses in in vitro functional assays. Notably, PRAME, a well-characterised cancer-testis antigen was confirmed as an H3K27M-associated immunogenic target, reinforcing its therapeutic relevance. Peptides identified exclusively in H3K27M+ cells were absent in KO models, demonstrating a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. Conclusions: This study provides the first systematic assessment of how H3K27M reshapes the antigenic landscape in DMG, uncovering novel, immunogenic tumour- associated peptides that could serve as targets for precision immunotherapy. By demonstrating that H3K27M mutation drives context-dependent antigen presentation, our findings establish a foundation for T cell-based therapies targeting H3K27M-associated antigens. These insights pave the way for next-generation personalised immunotherapies for this otherwise treatment-refractory disease. Key Points H3K27M mutation induces expression of tumour-associated antigens in DMG H3K27M alters the DMG immunopeptidome without uniformly changing HLA-I levels PRAME- and CBX2-derived peptides are immunogenic and targetable by CD8⁺ T cells Importance of Study Diffuse midline gliomas (DMGs) are universally fatal paediatric brain tumours with limited treatment options and poor immune visibility. While the H3K27M mutation is a defining hallmark, its impact on tumour immunogenicity remains unclear. This study presents the first comprehensive to explore the effect of H3K27M-mution on the DMG immunopeptidome, revealing six immunogenic peptides derived from epigenetically dysregulated tumour- associated antigens, including SLITRK2, PRAME, XKR5, and CBX2. These antigens elicited CD8⁺ T cell responses, establishing a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. By leveraging these altered antigens, we highlight actionable vulnerabilities for T cell-based immunotherapy.

Microgravity presents unique challenges to human physiology, particularly the immune system, during spaceflight. T lymphocytes, key components of adaptive immunity, play a vital role in immune regulation, yet the effects of microgravity on T-cell function and gene expression remain incompletely understood. This study, conducted as part of the MESSAGE (Microgravity Associated Genetics) Science Mission in the Axiom-3 mission ( https://www.nasa.gov/mission/station/research-explorer/investigation/?#id=9100 ). We aimed to investigate how microgravity influences immune cell responses by analysing blood samples from three astronauts between pre-flight and during their space mission on the ISS (post-launch Day 4, Day 7, and Day 10 in ISS). Hemogram analysis revealed no statistically significant differences in leukocyte, erythrocyte, haemoglobin, and haematocrit levels between pre-flight and in-flight samples, suggesting a stable haematological profile under microgravity conditions. T cell subpopulation analysis indicated fluctuations in effector memory T cells (TemEARLY and TemLATE), but these changes did not reach statistical significance. These findings suggest that, within the short duration of spaceflight, haematological and immune parameters remain largely stable, underscoring the need for further research into long-term immune adaptations in microgravity. As a next step, transcriptomic analysis will be performed to identify microgravity-associated gene candidates, and CRISPR-based knock-out of the genes in T cells will be generated to explore their functional roles in microgravity-induced immune modulation.

During their vectorial biosynthesis on the ribosome, elongating nascent polypeptide chains explore a range of conformational states towards their biologically functional structure. However, this high structural heterogeneity has limited their observation at high-resolution. Here, we have used an integrated structural biology approach to explore the structures of the multi-domain immunoglobulin-like FLN5-6 during its biosynthesis, capturing early folding through to native folding. We developed an in-silico purification approach for cryo-EM of ribosome-nascent chain complexes (RNCs), and integrated the resulting cryo-EM maps with NMR spectroscopy and atomistic molecular dynamics (MD) simulations to produce experimentally reweighted structural ensembles of RNC. The resulting atomistic structures reveal insights into the orientational heterogeneity of the nascent chain and its dynamic interactions with the ribosome. In particular, we find that two distinct pathways exist for nascent polypeptides in the exit tunnel vestibule, influenced by their stage of biosynthesis, folding conformational state and ribosomal RNA helices lining the tunnel. Our systematic analysis of the structures of nascent proteins translation-stalled at multiple time-points provides insights into how the ribosome dynamically modulates its pathway out of the exit tunnel to regulate its folding and accessibility for auxiliary factors of other co-translational events.

ABSTRACT Human pluripotent stem cell (hPSC)-derived cardiac therapies hold great promise for heart regeneration but face major translational barriers due to allogeneic immune rejection. Here, we engineered hypoimmunogenic hPSCs using a two-step CRISPR-Cas9 strategy: (1) B2M knockout, eliminating HLA class I surface expression, and (2) knock-in of HLA-E or HLA-G trimer constructs in the AAVS1 safe harbor locus to confer robust immune evasion. Hypoimmunogenic hPSCs maintained pluripotency, efficiently differentiated into cardiac cell types that resisted both T and NK cell-mediated cytotoxicity in vitro , and self-assembled into engineered cardiac organoids. Comprehensive analyses of the hypoimmunogenic cells and organoids revealed preservation of transcriptomic, structural, and functional properties with minimal off-target effects from gene editing. In vivo , hypoimmunogenic cardiac organoids restored contractile function in infarcted rat hearts and demonstrated superior graft retention and immune evasion in humanized mice compared to wild-type counterparts. These findings establish the therapeutic potential of hypoimmunogenic hPSC-CMs in the cardiac organoid platform, laying the foundation for off-the-shelf cardiac cell therapies to treat cardiovascular disease, the leading cause of death worldwide.

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 is evolutionarily conserved, yet subtle changes in its regulatory network drive species-specific traits. Transposable elements (TEs) contribute to genome evolution, but their role in cranial neural crest cells (CNCCs) remains unclear. Here, we investigate the domestication of hominoid-specific TEs (LTR5Hs and SVAs) as enhancers during human CNCC specification, a process critical for vertebrate craniofacial development. Using human iPSC-derived CNCCs, we identified ∼550 hominoid-specific TEs functioning as enhancers, including ∼250 human-specific, predominantly LTR5Hs. These elements are enriched for CNCC coordinator motifs, are bound by the CNCC signature factor TWIST1, and their enhancer activity appears largely CNCC-specific. CRISPR-interference targeting ∼75% of these active TEs led to widespread transcriptional dysregulation of genes involved in neural crest migration, and two orthogonal functional assays confirmed that CNCC migration is impaired upon TE repression. Finally, genes near human-specific TEs showed higher expression in human CNCCs compared to chimpanzee CNCCs, but TE repression restored gene expression to chimpanzee levels. These findings highlight how young TEs were domesticated to fine-tune CNCC regulatory networks, potentially contributing to lineage-specific craniofacial evolution.