CRISPR–Cas systems have revolutionized genome editing with their precision and versatility, enabling transformative applications in various fields, especially in the treatment of genetic diseases. However, the clinical translation of this technology is hindered by challenges such as off-target effects and uncontrolled nuclease activity. At the same time, it has the possibility of causing biosecurity risks, underscoring the urgent need for reliable regulatory tools. Existing CRISPR inhibitors, primarily anti-CRISPR protein or exogenously synthesized small molecules, are limited by their specificity or bioavailability and long research period, unable to address the diverse CRISPR nucleases used in research and therapy. Based on the phenomena obtained from various in vitro and cell experiments, combining molecular dynamics simulation and bio - layer interferometry (BLI) analysis, here we report a naturally occurring small-molecule β-nicotinamide mononucleotide (NMN), the first known endogenous metabolite with broad-spectrum inhibitory activity against multiple CRISPR-associated proteins (Cas9, Cas12, and Cas13) through various mechanisms. Our findings establish NMN as a dual-purpose tool, which reduces cell damage caused by gene editing and mitigates risks of unintended genetic modifications in research and clinical settings. This discovery further shortens the distance between basic medicine and translational medicine, providing a new approach for developing endogenous regulatory molecules in genome engineering.

Motivation Understanding double-strand break (DSB) repair and its disruption is key to decipher genomic instability driving diseases such as cancer and reveal therapeutic avenues. Numerous genes have been linked to DSB repair for the first time in recent genome-wide perturbation studies assessing effects on mutational outcomes. However, the functional roles of most such genes remain poorly understood. Evidence from other studies shows that related genes similarly modulate the frequency of specific mutational outcomes following DSB repair, but analysis has largely ignored the multiplicity of gene functions and cross-talk between pathways. Here, we infer functional roles for candidate genes based on knockout mutational spectra by identifying mutational signatures shared with known genes and then mapping them to DSB repair functions. Signatures are identified using non-negative matrix factorization (NMF) to reflect functional multiplicity and cross-talk. Results We generated mutational spectra for mouse embryonic stem (mES) cells at three target sites across CRISPR knockouts of 307 known and 459 candidate DSB repair genes. Analysis using NMF revealed four mutational signatures associated with homology-directed repair (HDR), microhomology-mediated end-joining (MMEJ), and the initiation and ligation components of non-homologous end-joining (NHEJ). Signatures suggested that candidate genes Dbr1 and Hnrnpk could influence MMEJ and Fanconi anaemia (FA), and that loss of core NHEJ components (e.g. MRN complex or Ku proteins) could shift repair preference towards Ku- independent NHEJ. These findings demonstrate the utility of NMF for characterizing the contribution of genes to repair pathways and provide a foundation to discover new gene functions in DSB repair. Availability github.com/joanagoncalveslab/MuSpectraNMF .

The fruit fly Drosophila is a powerful model organism for many biological questions. While Drosophila melanogaster has been the most widely used species owing to its versatile genetic tools, comparative studies also take advantage of the rich evolutionary, ecological, and behavioural diversity of Drosophilidae. This model system has only benefitted from the sequencing of hundreds of genomes assembled to chromosome level, and rich array of publicly-available datasets. However, labs that typically use just D. melanogaster can have difficulties adopting other species into their experiments due to unique dietary needs and care regimens. While dedicated research groups can rear dozens of fly species, this often requires preparing a variety of food media, which can be logistically taxing. In this study, we report an agar food recipe that we have used to rear >70 species of Drosophilidae (Prop recipe). This list includes ecological specialists such as D. sechellia , major pest species including D. suzukii , cactophiles, slime flux feeders, detritivores, and even mushroom-feeding species of the Quinaria and Testacea groups. This food recipe uses only standard powdered ingredients and molasses, and is already in use by one commercial transgenic injection provider. We provide life history parameters for dozens of species reared on this recipe, including comparisons in a subset of species across a variety of diets, finding fly fitness to be comparable or better on the Prop recipe among tested species. We further provide comparative data for 42 species after systemic infection by Acetobacter bacteria. However, unexpectedly, we found a striking interaction of diet and the microbiota that impacts susceptibility to infection in species-specific fashion. Our results suggest certain experiment types may be impacted by diet due to the microbiome that the diet itself supports. By providing rearing advice, life history parameters, and this infection dataset and experience, we hope to enable researchers to take advantage of the diversity of Drosophila as a model system for evolutionary research.

Resistance to androgen receptor (AR)-targeted therapies such as enzalutamide in castration-resistant prostate cancer (CRPC) often arises through lineage plasticity, yet the molecular mechanisms that define this process remain incompletely understood. While previous studies reported that Notch1 and Notch2 exert distinct and sometimes opposing effects in prostate cancer differentiation, the integrated role of Notch pathway activity has not been systematically explored. Here, we identify Notch signaling as a graded Rheostat that governs prostate cancer cell fate transitions. Integrative transcriptomic and functional analyses revealed that intermediate Notch activity maintains a stem-like progenitor state, whereas reduced or elevated signaling drives divergent differentiation trajectories toward luminal or neuroendocrine lineages, respectively. During CRPC progression and enzalutamide resistance, Notch signaling becomes dynamically rewired, peaking in progenitor-like populations that sustain plasticity and survival. Both CRISPR-mediated knockout and pharmacologic inhibition of Notch signaling depleted these progenitor cells and restored enzalutamide sensitivity. These findings demonstrate that the level, rather than the binary presence, of Notch signaling dictates lineage directionality and therapeutic response in CRPC, establishing it as a tunable and actionable driver of resistance.

Colorectal carcinoma (CRC) exerts a growing global disease burden, with microsatellite-stable/proficient mismatch repair (MSS/pMMR) tumors exhibiting intrinsic refractoriness to immune-checkpoint blockade (ICB) owing to low tumor mutational burden, limited neoantigenicity, and an immunosuppressive tumor microenvironment (TME) dominated by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). This review evaluates induced pluripotent stem cell (iPSC)–derived polyvalent vaccines as ontogenetically recapitulative immunogens capable of reinstating broad antitumor immunity. Reprogramming induces re-expression of oncofetal tumor-associated antigens, including cancer-testis antigens (NY-ESO-1, MAGE-A3), aberrant glycoforms of CEA and MUC1, and clinically actionable neoepitopes such as KRAS^G12D/V, thereby promoting epitope spreading and immunogenic cell death. Irradiated autologous or syngeneic iPSCs, delivered with Toll-like receptor 9 agonists, facilitate robust MHC I/II cross-presentation, driving CD8⁺ cytotoxic T-cell activation, Th1 polarization, perforin/granzyme-mediated cytolysis, and favorable effector-to-suppressor ratios. Preclinical models of melanoma, pancreatic ductal adenocarcinoma, and MSS CRC demonstrate prophylactic and therapeutic efficacy, with neoantigen-enhanced iPSCs synergizing with radiotherapy-induced DAMPs to achieve durable regressions and memory T-cell formation. Translational priorities include CRISPR-engineered hypoimmunogenic iPSC platforms, GMP-compatible non-integrating reprogramming, and combinatorial integration with STING agonists, ICB, CAR-NK cells, and LNP-mRNA constructs to enable biomarker-guided clinical deployment in minimal-residual-disease CRC.

Alternative splicing creates diverse RNA isoforms from individual genes, yet single-cell CRISPR screens are limited to gene-level quantification and cannot detect changes in alternative splicing and transcript isoforms. To overcome this limitation, we develop CRISPore-seq, which couples massively-parallel CRISPR perturbations with joint short- and long-read transcriptomics. CRISPore-seq simultaneously captures genetic perturbations and expression of genes, full-length transcripts and surface proteins in single cells. CRISPore-seq long reads identify 80% more transcript isoforms than short reads. Nearly all long reads map to unique transcript isoforms — in contrast to existing single-cell perturbation methods, which rarely distinguish specific isoforms. Using CRISPore-seq, we knock-down 15 different RNA-binding proteins (RBPs) and identify thousands of perturbation-driven alternative splicing events (ASEs). We find that exon skipping is the most common ASE observed and that skipped exons are enriched for binding sites of perturbed RBPs. Loss of the Nager syndrome-associated spliceosomal factor SF3B4 triggers skipping of exon 2 in the cell-cycle regulator CCND1 , preventing formation of a complex with CDK6 and blocking the G1-S transition. After rescue with a CCND1 isoform containing the skipped exon, both holoenzyme complex formation and cell proliferation are restored. By linking genes to transcriptional phenotypes with isoform-level resolution, CRISPore-seq is a highly scalable tool for understanding the impact of genetic perturbations on the human transcriptome.

Regulatory networks coordinate metabolism to control how plants adapt to biotic and abiotic stresses. This coordination can align transcriptional shifts across metabolic pathways using cis-regulatory elements shared across the enzyme genes within these pathways. While the role of transcription factors (TFs) in controlling this process across pathways is well known, less is known regarding the role of shared cis-regulatory elements across the genes in a pathway. Sharing cis-regulatory elements across the genes in an enzyme complex or pathway, can create coordinated regulation of the pathway by a TF. However, it is unclear if all the genes in a pathway or enzyme complex need to be fully coordinated for maximal function. For example, if one gene in an enzyme complex loses a cis-regulatory element, it may not alter the function of the enzyme complexes function if post-transcriptional or compensatory transcriptional changes are sufficient to balance the complex. To test how cis-modular membership shapes the function of an enzyme complex, we used CRISPR/Cas9 to abolish a common cis-regulatory element across the promoters of nine genes required for the mitochondrial pyruvate dehydrogenase complex (mtPDC). This complex is composed of three apoenzymes and is a central hub coordinating carbon flow between glycolysis and the tricarboxylic acid (TCA) cycle. Different combinations of these cis-element mutations were tested across the genes in the complex in Arabidopsis thaliana and the created genotypes were phenotyped for altered enzyme function using digital growth analysis, disease assays, metabolomics, and transcriptomics. This analysis revealed that mutating cis-element motifs of genes in this enzyme complex produced distinct phenotypes, displaying promoter-specific buffering and modularity.

Abstract Background Currently, surgical treatment options for pigment loss disorders are well-established. Studies have shown that melanocyte transplantation or melanin transplantation can yield favorable outcomes. However, this approach has not been widely adopted. The reasons for this may include insufficient sources of melanocytes, the lengthy process of extracting autologous melanocytes, and the high costs associated with their cultivation. Objective To improve the isolation of highly proliferative melanocytes, we seek to identify surface markers for selecting those with robust proliferative and differentiation potential. Methods Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) technology to label microphthalmia-associated transcription factor (MITF), induced melanocytes are obtained by differentiating pluripotent stem cells, and the proliferative capacity of different cell populations is assessed through a single-cell colony formation assay. Results This study verified that MITF-positive cells possess high proliferative capacity and consequently identified KIT proto-oncogene, receptor tyrosine kinase (KIT, CD117) as a characteristic surface marker. Conclusion The use of KIT allows for the isolation of induced melanocytes with high proliferative capacity, thereby improving production efficiency, though it may also lead to the loss of some highly proliferative cell subpopulations.

Wnt signaling controls embryonic development and tissue maintenance. Endocytosis of Wnt-receptors is required for signalling, yet uptake mechanisms remain poorly understood. Here, we identify the actin regulator and Ena/VASP protein, Mena, as a key mediator. Upon Wnt stimulation, Mena redistributes from focal adhesions to signalosomes, Wnt-receptor clusters. Mena directly binds Wnt-coreceptors LRP5/6 in a phosphorylation-dependent manner increasing Wnt signal transduction. Sequestration of Ena/VASP proteins impedes in vivo Wnt activation driving Xenopus embryonic development. We resolve previous controversies by showing that Wnt3a triggers rapid Clathrin-Mediated and Fast Endophilin-Mediated LRP6 endocytosis at low concentrations sufficient for Wnt activation. This efficient endocytosis requires Ena/VASP proteins and is specifically promoted by Mena. Our results suggest Mena as a crucial mediator of Wnt signalosome endocytosis thus promoting canonical Wnt signalling.

This conceptual manuscript delineates an interdisciplinary framework for ``The Divine Gift'' (TDG), a hypothetical bio-nano-programmed therapeutic agent designed to target biological pathogens, correct genetic and regulatory dysfunctions, and mitigate pathological aging in humans. Integrating established principles from medicine, pharmacology, biochemistry, nanotechnology, and systems biology, the framework explores potential mechanisms for healthspan extension. Mathematical models, derivations, and simulations are detailed, drawing analogies from epidemiological and genomic databases. Sensitivity analyses, statistical methods, Bayesian inference, uncertainty quantification, and falsifiability criteria underpin methodological rigor and reproducibility. References are sourced from peer-reviewed literature with verifiable identifiers. The framework includes biochemical schematics, manufacturing outlines, and ethical considerations. As a theoretical construct, TDG addresses implications for overpopulation and socioeconomic dynamics, proposing open-source strategies for equitable access. Expanded discussions on CRISPR applications in agriculture, veterinary medicine, and human therapeutics provide insights into scalability and translational potential. Originality is ensured through refined conceptual integration.

Background: Infections caused by the multidrug-resistant pathogen Mycobacterium abscessus (Mab) are notoriously difficult to treat. The novel β-lactamase inhibitor durlobactam, in combination with β-lactams, shows potent bactericidal activity against Mab, but the potential for acquired resistance remains a clinical concern. Objectives: To identify and characterize mechanisms of acquired resistance to durlobactam in Mab. Methods: In vitro single-step resistance selection was performed by plating wild-type Mab ATCC 19977 on agar containing durlobactam. Resistant mutants were isolated, and their genomes were sequenced. The resistance phenotype was confirmed by constructing a targeted gene deletion mutant and by transcriptional silencing using a CRISPR interference (CRISPRi) system. Minimum inhibitory concentrations (MICs) were determined by both, an agar-based method and broth microdilution. Results: Whole-genome sequencing of durlobactam-resistant mutants identified loss-of-function mutations in ponA1, a gene encoding a class A penicillin-binding protein involved in cell wall synthesis. Targeted deletion of ponA1 (ΔponA1) and CRISPRi-mediated knockdown of ponA1 expression both recapitulated the resistance phenotype, resulting in a significant increase in the durlobactam MIC on solid agar media. Strikingly, broth microdilution MICs remained largely unaffected. Conclusions: Inactivation of the peptidoglycan synthase PonA1 is a novel mechanism of resistance to durlobactam in Mab that is phenotypically expressed only during growth on solid surfaces. This finding identifies a specific genetic pathway for resistance and highlights that standard broth-based susceptibility testing could miss clinically relevant resistance mechanisms.

Metastatic breast cancer (MBC) remains a formidable clinical challenge due to its aggressive nature, genetic heterogeneity, and limited treatment success. Traditional pre-clinical models, including two-dimensional (2D) cell cultures and animal models, often fall short in accurately replicating the complex human tumor microenvironment (TME) and predicting clinical outcomes. This inadequacy has driven the urgent development of advanced non-animal models. This report details the capabilities of three-dimensional (3D) cell cultures, patient-derived organoids (PDOs), and organ-on-a-chip (OoC) systems as leading non-animal platforms. These innovative models offer enhanced physiological relevance, faithfully mimic tumor heterogeneity, and integrate critical TME components, providing a more reliable basis for studying the chemotherapeutic effects of drugs on breast cancer metastasis. Furthermore, the integration of emerging technologies like 3D bioprinting, CRISPR/Cas9 genome editing, advanced imaging, and artificial intelligence (AI), coupled with collaborative consortia, is poised to revolutionize personalized medicine and accelerate drug discovery, ultimately reducing reliance on animal testing and improving patient outcomes.

Lignin, a complex natural aromatic polymer, poses significant challenges to its efficient degradation, hindering the utilization of biomass for many industrial applications. Bacterial degradation of lignin may offer a promising solution to this challenge. This project aimed at elucidating the function of secreted oxidative enzymes from Pseudomonas putida involved in lignin degradation and utilization. Using CRISPR-Cas9 and CRISPR-Cas3 systems, the putative lignin-degrading versatile peroxidase gene (VP; PP _ 1686 , originally annotated as glutathione peroxidase GPx) and dye-decolorizing peroxidase gene ( PP_3248 ) were individually knocked out from P. putida KT2440. The ΔPP_1686 mutant exhibited impaired growth and utilization of lignin-derived compounds. This correlated with reduced expression of p-hydroxybenzoate hydroxylase pobA and of DNA repair modules, alongside compensatory upregulation of energy and redox supply pathways. This work expands our knowledge on bacterial glutathione peroxidase by presenting a role beyond ROS scavenging. This work revealed the importance of P. putida VP/GPx in maintaining redox balance while supporting lignin-derived aromatic metabolism, offering new targets for future investigation into stress–metabolism crosstalk and lignin valorization strategies.

Bacteriophages have genomes that span a wide size range, are densely packed with coding sequences, and frequently encode genes of unknown function. Classical forward genetics has defined essential genes for phage replication in a few model systems but remains laborious and non-scalable. Unbiased functional genomics approaches are therefore needed for phages, particularly for large lytic phages. Here, we develop a phage transposon sequencing (TnSeq) platform that uses the mariner transposase to insert an anti-CRISPR selectable marker into phage genomes. CRISPR-Cas13a–based enrichment of transposed phages followed by pooled sequencing identifies both fitness-conferring and dispensable genes. Using the Pseudomonas aeruginosa -infecting nucleus-forming jumbo phage ΦKZ (280,334 bp; 371 predicted genes) as a model, we show that ∼110 genes are fitness-conferring via phage TnSeq. These include conserved essential genes involved in phage nucleus formation, protein trafficking, transcription, DNA replication, and virion assembly. We also isolate hundreds of individual phages with insertions in non-essential genes and reveal conditionally essential genes that are specifically required in clinical isolates, at environmental temperature, or in the presence of a defensive nuclease. Phage TnSeq is a facile, scalable technology that can define essential phage genes and generate knockouts in all non-essential genes in a single experiment, enabling conditional genetic screens in phages and providing a broadly applicable resource for phage functional genomics.

Abstract Objective Human endogenous retroviruses (HERVs) have been the focus of numerous recent studies. HERVs entered the human genome millions of years ago and are associated with various diseases, including cancer and immune regulation. Among them, the HERV-K family exhibits the highest transcriptional activity. However, little is known about the expression of HERVs in acute myeloid leukemia (AML) or their potential as biomarkers and therapeutic targets. This study primarily investigated the role of HERV-K102 in the development of AML and explored its underlying mechanisms. Methods The expression profiles of HERV K102 in AML and normal samples were analyzed using The Cancer Genome Atlas (TCGA) database and AML cell lines. Knockout models were generated through CRISPR-Cas9-mediated deletion of the HERV-K102 envelope (K-ENV). Cell viability and pyroptosis rates were measured using the CCK-8 assay and flow cytometry, respectively. Transcriptome analysis was performed to identify differentially expressed genes and related pathways. Western blotting and detection of pyroptosis markers were conducted. Furthermore, the role of HERV-K102 in AML was validated in an inducible knockout xenograft tumor model. Results HERV-K102 was aberrantly activated and highly expressed in AML, and its expression correlated with poor prognosis. K-ENV depletion inhibited AML cell proliferation and promoted apoptosis. Moreover, K-ENV knockout induced pyroptosis, as indicated by increased lactate dehydrogenase (LDH) release, enhanced cleavage of caspase-1 and gasdermin D (GSDMD), and characteristic morphological features of pyroptotic cells. Mechanistically, transcriptomic and functional analyses demonstrated that this process was mediated by S100A9 upregulation and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome pathway. Conclusions Our findings suggest that HERV-K102 ENV plays a critical role in AML pathogenesis and may represent a novel diagnostic and therapeutic target.

Abstract Background Human T-lymphotropic virus type 1 (HTLV-1) infects up to ten million people worldwide, and causes severe diseases, including adult T-cell leukemia/lymphoma and HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP). Individuals with HAM/TSP are prone to pulmonary complications (e.g., bronchiectasis). Their bronchoalveolar lavage fluid typically shows increased levels of inflammatory cytokines, chemokines and cell adhesion molecules contributing to chronic inflammation. Results This study assessed the impact of HTLV-1 infection on lung inflammation by analyzing the alveolar transcriptome of A549 epithelial cells following exposure to HTLV-1. Co-culture with HTLV-1-infected MT-2 cells caused transcriptomic changes related to viral response, NF-κB activation, and inflammation. RT-qPCR confirmed elevated expression of the chemokine monocyte chemotactic protein-1 (MCP-1/CCL2) and colony stimulating factor 1 (CSF-1) in A549 MT-2 co-cultures. Increased CSF-1 expression was mechanistically linked to NF-κB signaling, using CRISPR/Cas9 RELA knockout. Supernatant from A549 MT-2 co-cultures triggered chemotaxis and macrophage differentiation of THP-1 and primary monocytes. Systems biology analysis revealed enrichment in pathways associated with monocyte infiltration and bronchiectasis. Finally, we validate the in vivo relevance of our in vitro model through multi-cohort multi-omics analysis combining bulk and single-cell transcriptomics, viral interactomics and multi-ancestry GWAS. Conclusions We describe an in vitro co-culture model that recapitulates HTLV-1-triggered lung inflammation, through RELA/NF-kB-dependent release of pro-inflammatory cytokines and chemokines resulting in monocyte chemotaxis, activation and differentiation. Integrated multi-omics analysis confirmed the in vivo relevance of our in vitro model.

Dissecting cell-state-specific changes in gene regulation following perturbations is crucial for understanding biological mechanisms. However, single-cell sequencing provides only unmatched snapshots of cells under different conditions. This destructive measurement process hinders the estimation of individualized treatment effects (ITEs), which are essential for pinpointing these heterogeneous mechanistic responses. We present scDRP, a generative framework that lever-ages disentangled representation learning to separate perturbation-dependent and perturbation-independent latent variables via a sparsity regularized β -VAE. Assuming quantile-preserving effects of perturbations conditional on confounders, scDRP performs conditional optimal transport in the latent space to infer counterfactual states and estimate ITEs. Applied to simulated and real single-cell perturbation datasets, scDRP accurately estimates treatment effects and individual counterfactual responses, revealing cell type-specific functional gene module dynamics. Specifically, it captures distinct cellular patterns under rhinovirus and cigarette-smoke extract exposures, reveals heterogeneous responses to interferon stimulation across diverse immune cell types and identified distinct functional module activation in chronic myeloid leukemia cells following CRISPR knockouts targeting different genes. scDRP also generalizes to unseen perturbation doses and combinations. Our framework provides a principled computational approach to elucidate heterogeneous causal relationships from single-cell perturbation data, promoting to a deeper understanding of cellular and molecular mechanisms.

ABSTRACT Gene expression regulation is a stochastic process that can be modified by mutations not only in a deterministic manner but also in a probabilistic way, for example by changing the extent of cell-to-cell variability in gene expression (also called “gene expression noise”) without necessarily changing the expected (mean) expression level. Such mutations can act either in cis (perturbing expression noise at their own locus) or in trans (perturbing expression noise of a gene located elsewhere in the genome). Although systematic studies have successfully analyzed the properties of cis -acting mutations modulating gene expression noise, less is known about the type of genetic changes that may alter expression noise in trans . Here, we applied genetic mapping on yeast strains ( Saccharomyces cerevisiae ) generated by random mutagenesis and identified three mutations (in chs1 , yme2 and msh1 genes) that changed the expression noise of a reporter gene in the nuclear genome regulated by the yeast TDH3 promoter. Each of these mutations affected either extrinsic noise (variability due to cell-specific factors), intrinsic noise (inherent variability within each cell), or both. Surprisingly, all three mutations targeted proteins located outside of the nucleus: Yme2 and Msh1 being involved in the maintenance of mitochondrial genome integrity, and Chs1 being necessary for the repair of cell-wall defects in freshly-born daughter cells. Our results reveal that mitochondrial state can modulate the extent of intrinsic expression noise of eukaryotic nuclear genes.

Activin-class ligands of the transforming growth factor β family induce follicle-stimulating hormone (FSH) production by pituitary gonadotrope cells in mice via the actions of the transcription factors SMAD3, SMAD4, and FOXL2, which bind to cis -elements in the FSHβ subunit ( Fshb ) promoter. An enhancer region for murine Fshb transcription was identified in vitro . However, deletion of the region using CRISPR-Cas9 did not affect FSH synthesis or secretion in mice. Using single-nucleus ATAC-seq of whole murine pituitaries, we identified three additional open chromatin regions upstream of Fshb exclusively in gonadotropes. These regions, as well as the Fshb gene, were fully or partially closed in gonadotropes of FSH-deficient mice with genetically or pharmacologically inactivated activin type II receptors. The initially characterized enhancer region did not significantly alter basal or activin-stimulated murine Fshb promoter-reporter activity in homologous LβT2 cells. In contrast, the other three open chromatin regions enhanced basal and activin A-stimulated Fshb promoter-reporter activity in LβT2 cells, with the two most distal showing the greatest effects. These two regions were open, exhibited enrichment of the enhancer mark H3K27ac, and were bound by SMAD2/3 and FOXL2 in response to activin A in LβT2 cells. The most distal enhancer exhibited strong FOXL2 and weak SMAD4 binding in gel shift assays. SMAD4, but not FOXL2, directly bound the other distal enhancer. Mutation of defined FOXL2 and SMAD4 cis -elements diminished enhancer activity in reporter assays in LβT2 cells. Collectively, the data indicate that there may be as many as four activin-sensitive enhancers upstream of murine Fshb .

Colocalisation analysis is extensively applied across diverse GWAS and molecular QTL datasets to identify candidate causal genes. We systematically characterised large-scale colocalisation results across eQTL studies varying in cellular granularity and sample size, with the goal of providing design and interpretation recommendations. We found 34-50% of GWAS hits colocalised, and were more likely to colocalise if they were located nearer genes and had a more common lead variant. We also found over 50% of colocalisations were found in only one cell type. This led to an inherent trade-off: while high granularity studies tended to have smaller sample sizes and lower eQTL discovery, each eQTL from these high-granularity datasets were more likely to colocalise, reflecting cell-type specificity. On the other hand, lower granularity studies achieved larger sample size and higher eQTL discovery, leading to detection of the greatest total number of colocalisations, particularly for lower frequency GWAS lead variants. This suggests large, high granularity studies will be needed to identify remaining colocalisations. Of the peaks that colocalised, 37-47% did so with multiple genes, suggesting coregulation of the GWAS trait, horizontal pleiotropy, or false positives. However, sensitivity analyses indicated that even extremely stringent significance thresholds did not substantially reduce multi-gene colocalisations, arguing against widespread false discovery. Integration of enhancer–promoter interaction data provided evidence for coregulation among multi-colocalising eGenes. While disentangling causality from horizontal pleiotropy will ultimately require experimental perturbation, triangulation using different sources of observational data is likely to be necessary, provided careful consideration is taken to identify biases and missing data that may influence gene prioritisation.

Alveolar rhabdomyosarcoma (RMS), an aggressive pediatric soft tissue cancer, is driven by the oncogenic fusion transcription factor PAX3::FOXO1 (P3F) or PAX7::FOXO1. In a subset of fusion-positive (FP)-RMS cases, amplification of the MIR17HG locus leads to overexpression of the miR-17-92 cluster of microRNAs (miRNAs). However, miR-17-92 is also highly expressed in FP-RMS tumors lacking this amplification, suggesting alternative regulatory mechanisms. Here, we show that P3F and MYCN cooperatively drive miR-17-92 expression in FP-RMS. CRISPR/Cas9-mediated knockout of P3F or MYCN in FP-RMS cell lines substantially reduced miR-17-92 expression. Using a human myoblast line or low P3F FP-RMS variant with inducible P3F or MYCN expression, P3F or MYCN alone induces minimal to low miR-17-92 expression whereas introduction of both MYCN and P3F leads to robust activation of the miR-17-92 cluster and acquisition of oncogenic phenotypes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed a P3F binding motif located 1.84 Mb upstream of the MIR17HG promoter. CRISPR-mediated deletion of this region in the myoblast system resulted in marked reduction of miR-17-92 expression and impaired oncogenic transformation. Functional inhibition of mature miRNAs of this cluster in FP-RMS cells using miRNA-sponge constructs suppressed proliferation and transformation. In the myoblast model system, transduction studies with exogenous miR-17-92 or miRNA-sponge expression constructs indicated that miR-17-92 is necessary but not sufficient for oncogenic transformation. Together, these findings establish a cooperative transcriptional axis in FP-RMS involving P3F and MYCN that activates MIR17HG through a distal regulatory element, thereby contributing to oncogenic behavior and uncovering a novel mechanistic vulnerability.

ABSTRACT Although the fundamental architecture of metazoan nervous systems is typically established in the embryo, substantial numbers of neurons are added during post-natal development while existing neurons expand in size, refine connectivity, and undergo additional differentiation. To reveal the underlying molecular determinants of post-embryonic neurogenesis and maturation, we have produced gene expression profiles of all neuron types and their progenitors in the first larval stage (L1) of C. elegans . Comparisons of the L1 profile to the embryo and to the later L4 larval stage identified thousands of differentially expressed genes across individual neurons throughout the nervous system. Key neuropeptide signaling networks, for example, are remodeled during larval development. Gene regulatory network analysis revealed potential transcription factors driving the temporal changes in gene expression across the nervous system, including a broad role for the heterochronic gene lin-14. We utilized available connectomic data of juvenile animals in combination with our neuron-specific atlas to identify potential molecular determinants of membrane contact and synaptic connectivity. These expression data are available through a user-friendly interface at CeNGEN.org for independent investigations of the maturation, connectivity and function of a developing nervous system.

Background Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 13a (Cas13a) has been described as a superior tool to short-interfering RNAs (siRNAs) for specific gene silencing. Cas13 targets RNAs through Watson-Crick binding of the target CRISPR RNA (crRNA) and activation of nuclease activity. In bacteria, once Leptotrichia wadei Cas13a (LwCas13a) has cut its specific target, the trans-collateral activity of the protein degrades any single-stranded RNA present in the cell independent of its sequence or homology to the crRNA. This transcollateral activity has been reported to be absent in mammalian cells. Therefore, in this study, we aimed to downregulate mRNAs expression in mammalian cells (HaCaT and HEK293T) using LwaCas13a. Methods We developed a doxycycline-inducible system to express LwaCas13a in HEK293T cells. The off-target activity of LwaCas13a in HEK293T cells was analyzed using RNA-seq. Results In this study, we observed that activation of LwaCas13a in HEK293T cells led to non-specific targeting of RNAs, which caused cell toxicity and death. Conclusion This study provides evidence of the off-target activity of LwaCas13a in HEK293T cells, making it an unsuitable tool for the specific downregulation of RNAs.

The development of complex organs, like the brain, demands a robust system for tissue remodeling and cellular debris clearance. In the brain, this function is performed by microglia, which must clear diverse debris substrates, including that caused by cell death. Although the subsequent fate of these phagocytic microglia is a critical regulatory point that impacts whether the brain resolves a debris environment, the genetic mechanisms that control microglia fate after debris clearance remain mostly unknown. To address this, we conducted a large-scale CRISPR screen in zebrafish using a custom-built robotic confocal microscope. We selected candidate genes from a single-cell RNA sequencing dataset of embryonic mouse microglia. This screen identified several modulators of microglial lifespan and cannibalism that are enriched in mouse and zebrafish microglia, including interleukin-10 receptor beta ( il10rb ), a receptor subunit for the cytokine IL10. Perturbation of il10 , il10rb, and downstream signaling molecules JAK/STAT in zebrafish reduced microglial death. Expression analysis in mouse and zebrafish confirmed that microglia express both il10 and il10rb . Given the established role of IL10 in lysosomal remodeling, we hypothesized that it regulates microglial survival through lysosomal acidification. While il10rb perturbation did not alter lysosome number or size, it caused a significant reduction in LysoTracker-positive lysosomes, indicating decreased lysosomal acidification. Inhibiting v-ATPase also reduced microglial death, reinforcing the link between lysosomal pH and cell fate. Our findings reveal a cytokine-regulated mechanism where lysosomal dynamics determine the survival of phagocytic microglia. We propose that a necroptosis-cannibalism process functions as a quality control mechanism for microglial turnover, which is critical for refining neuroimmune cell function in the brain.

Mutation specific therapeutic approaches, like exon skipping or gene-editing, hold promise for the treatment of Duchenne muscular dystrophy (DMD). Translatability of preclinical studies investigating these approaches could greatly be improved through the use of humanized mouse models, as these allow preclinical testing of human specific sequences. We developed four novel humanized DMD mouse models with either a deletion of exon 44, 45, 51 or 53 in the human DMD gene, in a mouse dystrophin negative background ( mdx mouse; exon 23 nonsense mutation). Our optimized prescreening pipeline allowed us to do so very efficiently with the CRISPR-Cas9 technology. We confirmed either complete lack of dystrophin, or expression of trace levels, which led to development of muscle pathology consisting of muscle fiber de-, and regeneration, inflammation and fibrosis in young adult mice. Intramuscular treatment with vivo-morpholinos targeting a flanking exon induced exon skipping in the DMD strains, which restored the disrupted open reading frame and subsequently dystrophin expression. This validates these models as valuable tools for preclinical studies investigating human sequence specific therapeutic approaches for DMD. Summary statement Humanized Duchenne muscular dystrophy mouse models were created with deletions of exon 44, 45, 51 or 53 in the human DMD gene. These dystrophic models allow preclinical testing of human-specific dystrophin restoring approaches.