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.

Current standard food detection methods do not distinguish between infectious and non-infectious norovirus leading to uncertainty in the interpretation and management of a norovirus positive food sample. These methods also require expensive RT-qPCR based equipment. In contrast, CRISPR-based, compared to RT-qPCR based, detection methods yield similar sensitivity and specificity and are generally less expensive. The aim of this study was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR-Cas13a based detection method in conjunction with acapsid integrity assay. Our CRISPR method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/ 25 g (5 gc/ rx). This method did not cross-react with other targets (synthetic hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, this CRISPR based method showed an increased sensitivity when detecting low copy numbers of RNase-pretreated MNV-1 in lettuce and blueberries samples. This is the first report describing a CRISPR-based detection of potentially infectious viruses in food samples.

The PI3K/AKT signaling pathway is frequently dysregulated in cancer and controls key cellular processes such as survival, proliferation, metabolism and growth. Protein glycosylation is essential for proper protein folding and is also often deregulated in cancer. Cancer cells depend on increased protein folding to sustain oncogene-driven proliferation rates. The N-glycosyltransferase asparagine-linked glycosylation 3 homolog (ALG3), a rate-limiting enzyme during glycan biosynthesis, catalyzes the addition of the first mannose to glycans in an alpha-1,3 linkage. Here we show that ALG3 is phosphorylated downstream of the PI3K/AKT pathway in both growth factor-stimulated cells and PI3K/AKT hyperactive cancer cells. AKT directly phosphorylates ALG3 in the amino terminal region at Ser11/Ser13. CRISPR/Cas9-mediated depletion of ALG3 leads to improper glycan formation and induction of endoplasmic reticulum stress, the unfolded protein response, and impaired cell proliferation. Phosphorylation of ALG3 at Ser11/Ser13 is required for glycosylation of cell surface receptors EGFR, HER3 and E-cadherin. These findings provide a direct link between PI3K/AKT signaling and protein glycosylation in cancer cells.

The leading region of conjugative plasmids is the first to enter recipient cells during conjugation. This region is enriched in anti-defence genes, suggesting that defence system evasion shapes plasmid organization. Might this selective pressure also variably impact sequence composition? Here we hypothesize that the leading region is subject to stronger selection to avoid triggering defence systems. We investigate this for Type II restriction-modification (RM) systems, using a dataset of thousands of conjugative plasmids belonging to different plasmid taxonomic units (PTUs). Consistent with our hypothesis, we find evidence of RM target depletion in leading regions, despite higher average GC-content in the leading region which should increase palindrome density. We find evidence of strand-specific nucleotide skews that could cause this depletion, which we suggest could have drivers beyond RM evasion. Our work opens up intriguing questions about how the sequence composition of the leading region has been shaped by different selective pressures.

First identified in 2009, Candidozyma auris (formerly Candida auris ) is an emerging multidrug resistant fungus that can cause invasive infections with a crude mortality rate ranging from 30-60%. Currently, 30-50% of C. auris isolates are intrinsically resistant to amphotericin B. In this work, we characterized a clinical case of acquired amphotericin B resistance using whole genome sequencing, a large-scale phenotypic screen, comprehensive sterol profiling, and genotypic reversion using CRISPR. Data obtained in this work provides evidence that a deletion resulting in a frameshift in ERG3 contributes to the observed resistant phenotype. Characterization of this isolate also revealed a fitness cost is associated with the abrogation of ergosterol production and its replacement with other late-stage sterols. This article presents a clinical case description of amphotericin B resistance from a frameshift mutation in ERG3 in C. auris and marks an advancement in the understanding of antifungal resistance in this fungal pathogen.

Gene discovery studies in individuals with diabetes diagnosed within 6 months of life (neonatal diabetes, NDM) can provide unique insights into the development and function of human pancreatic beta-cells. We describe the identification of homozygous PAX4 loss-of-function variants in 2 unrelated individuals with NDM: a p.(Arg126*) stop-gain variant and a c.-352_104del deletion affecting the first 4 PAX4 exons. We confirmed the p.(Arg126*) variant causes nonsense mediated decay in CRISPR-edited human induced pluripotent stem cell (iPSC)-derived pancreatic endoderm cells. Integrated analysis of CUT&RUN and RNA-sequencing in PAX4 -depleted islet cell models identified genes directly regulated by PAX4 involved in both pancreatic islet development and glucose-stimulated insulin secretion. Both probands had transient NDM which remitted in early infancy but relapsed between the ages of 2 and 7 years, demonstrating that in contrast to mouse models, PAX4 is not essential for the development of human pancreatic beta-cells.

CAR-T cell therapies are revolutionizing the treatment of refractory and relapsed haematological malignancies, but many patients do not exhibit long-term responses, and these therapies are less effective against solid tumors. Poor persistence of CAR-T cells in patients is associated with therapeutic failure, highlighting the need to identify strategies promoting in vivo expansion. Here, we developed an in vivo competitive screening method to identify genes whose inactivation confers a selective advantage to CAR-T cells. Inactivation of 50 genes in a heterogeneous population of T cells expressing an EGFR-targeting CAR revealed that disruption of REGNASE-1, SOCS1, PTPN2, and P16INK4A conferred a selective advantage to CAR-T cells in human lung tumor-bearing mice. Consistently, inactivation of these genes improved tumor eradication by CAR-T cells. Interestingly disruption of other genes, described to improve CAR-T cell function in other contexts, had a negative impact in this orthotopic lung tumor model. Further evaluation of long-term effects in a subcutaneous model, highlighted SOCS1 ablation as the most promising strategy for in vivo CAR-T cell amelioration. These results support the importance of evaluating CAR-T cell editing strategies in tumor-specific models and highlight the versatility of our screening approach as a pre-clinical tool for context-specific studies on CAR-T cells amelioration.

Cell migration plays a key role in normal developmental programs and in disease, including immune responses, tissue repair, and metastasis. Unlike other cell functions, such as proliferation which can be studied using high-throughput assays, cell migration requires more sophisticated instruments and analysis, which decreases throughput and has led to more limited mechanistic advances in our understanding of cell migration. Current assays either preclude single-cell level analysis, require tedious manual tracking, or use fluorescently labeled cells, which greatly limit the number of extracellular conditions and molecular manipulations that can be studied in a reasonable amount of time. Using the migration of cancer cells as a testbed, we established a workflow that images large numbers of cells in real time, using a 96-well plate format. We developed and validated a machine-vision and deep-learning analysis method, DeepBIT, to automatically detect and track the migration of individual cells from time-lapsed videos without cell labeling and user bias. We demonstrate that our assay can examine cancer cell motility behavior in many conditions, using different small-molecule inhibitors of known and potential regulators of migration, different extracellular conditions such as different contents in extracellular matrix and growth factors, and different CRISPR-mediated knockouts. About 1500 cells per well were tracked in 840 different conditions, for a total of ~1.3M tracked cells, in 70h (5 min per condition). Manual tracking of these cells by a trained user would take ~5.5 years. This demonstration reveals previously unidentified molecular regulators of cancer cell migration and suggests that collagen content can change the sign of how cytoskeletal molecules can regulate cell migration.

Stem cell populations in tissues require precise regulation of their number and quality to maintain proper organ growth and regenerative capacity. Amongst various regulatory mechanisms, immune cells are emerging to directly regulate stem cell populations. The medaka retinal stem cell niche, a model for lifelong neurogenic growth, provides a system to study immune cell-stem cell interactions. Here we investigate how microglia, the resident macrophages of the central nervous system, regulate the retinal stem cell niche. We identify that bona fide retinal stem cells express the chemokine ccl25b while its cognate receptor, ccr9a, is expressed in microglia. These microglia form a static surveillance ring adjacent to the stem cell niche and actively phagocytose retinal stem cells. We show that targeted mutation of ccl25b affects microglia mobility and leads to reduction of retinal-stem cell-derived material in microglia. Interference with microglia by deletion of spi1b reveals that microglia absence leads to increased numbers of ccl25b-positive stem cells and results in morphological defects in the retinal stem cell niche and retina. Overall, our data show that under homeostatic conditions the retinal stem cell population, essential for proper eye development, is actively pruned by immune surveillance.

Centromeres in eukaryotes are defined by the presence of histone H3 variant CENP-A/CENH3. Chlamydomonas encodes two predicted CENH3 paralogs, CENH3.1 and CENH3.2, that have not been previously characterized. We generated peptide antibodies to unique N-terminal epitopes for each of the two predicted Chlamydomonas CENH3 paralogs as well as an antibody against a shared CENH3 epitope. All three CENH3 antibodies recognized proteins of the expected size on immunoblots and had punctuated nuclear immunofluorescence staining patterns. These results are consistent with both paralogs being expressed and localized to centromeres. CRISPR-Cas9 mediated insertional mutagenesis was used to generate predicted null mutations in either CENH3.1 or CENH3.2 . Single mutants were viable but cenh3.1 cenh3.2 double mutants were not recovered, confirming that the function of CENH3 is essential. We sequenced and assembled two chromosome-scale Chlamydomonas genomes from strains CC-400 and UL-1690 (a derivative of CC-1690) with complete centromere sequences for 17/17 and 14/17 chromosomes respectively, enabling us to compare centromere evolution across four isolates with near complete assemblies. These data revealed significant changes across isolates between homologous centromeres including mobility and degeneration of ZeppL-LINE1 (ZeppL) transposons that comprise the major centromere repeat sequence in Chlamydomonas. We used Cleavage Under Targets and Tagmentation (CUT&Tag) to purify and map CENH3-bound genomic sequences and found enrichment of CENH3-binding almost exclusively at predicted centromere regions. An interesting exception was chromosome 2 in UL-1690, which had enrichment at its genetically mapped centromere repeat region as well as a second, distal location, centered around a single recently acquired ZeppL insertion. The CENH3-bound regions of the 17 Chlamydomonas centromeres ranged from 63.5kb (average lower estimate) to 175kb (average upper estimate). The relatively small size of its centromeres suggest that Chlamydomonas may be a useful organism for testing and deploying artificial chromosome technologies.

CEP290 is an important human disease gene, as mutations are implicated in a broad spectrum of autosomal recessive ciliopathies, including Leber congenital amaurosis and Joubert, Meckel, Senior-LØken or Bardet Biedl syndromes. To create isogenic mutant human induced pluripotent stem cell (hiPSC) lines for disease modeling, we employed CRISPR/Cas9 to introduce disease-relevant mutations into the control hiPSC line HMGU1 (ISFi001-A). Thorough characterization of the lines, including the effect of the mutation at the mRNA and protein level, shows that these CEP290 -mutant lines provide a useful resource for studying ciliopathy disease mechanisms and cilia biology through differentiation into diverse cell types and organoids.

CRISPR–Cas systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR–Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56,343 CRISPR arrays across 25,628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting spacer conservation. This observation corroborates recent findings by Fehrenbach et al. (2024) indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.

Colorectal cancer (CRC) remains a major global health concern, partly due to resistance to therapy and the lack of new effective treatments for advanced disease. The combination of 5-Fluorouracil (5FU, a thymidylate synthase inhibitor) and irinotecan (a topoisomerase 1 inhibitor) is widely used in first-line and subsequent treatments. This study aimed to identify novel therapeutic targets to enhance combinatorial therapy, improving treatment efficacy and durability of response. We performed a loss-of-function screen using HT29 CRC cell line and a retroviral library containing 7296 shRNAs targeting 912 chromatin genes. Cells were then treated with 5FU and SN38 (the active metabolite of irinotecan) or left untreated for 4 weeks. Genes enriched in resistant clones were identified through next-generation sequencing. Among candidate genes, PARG was selected for functional validation. CRISPR/Cas9-mediated knockout (HT29 PARG-KO) resulted in increased global poly(ADP-ribosyl)ation after 5FU and SN38 treatment. PARG depletion led to reduced cell viability and increased apoptosis, particularly after 5FU exposure. Pharmacological PARG inhibition (PDD00017273) synergized with 5FU and SN38 across three CRC models (HT29, DLD1, HT115). In vivo , HT29 PARG-KO xenografts were more sensitive to 5FU. Immunohistochemical analysis of 170 CRC patient tumors revealed that positive PARG expression correlated with poor response to 5FU + Irinotecan, increased liver metastases, and worse long-term survival. Our findings highlight PARG as a promising therapeutic target for CRC, where its inhibition enhances the efficacy of standard chemotherapy.