ABSTRACT Imaging-based CRISPR screens enable high-content functional genomics by capturing phenotypic changes in cells after genetic perturbation. Protein barcodes provide cost-effective, easy-to-implement, and imaging-compatible barcoding for pooled perturbations, yet their scalability has been constrained by the need for arrayed cloning, lentiviral recombination between barcodes and guides, and difficulties in decoding barcodes with high confidence. Here, we introduce poolVis and cellPool, an integrated experimental and computational platform designed to address these limitations. poolVis uses Cre-lox-mediated reconfiguration to position barcode-sgRNA pairs in proximity during viral integration, which greatly reduces barcode shuffling during pooled cloning and delivery. cellPool leverages a scalable computational workflow and the unique aspects of protein barcodes to produce unpooled image galleries from multi-terabyte scale datasets. Applying this platform to single- and double-CRISPRi profiling of cell-cycle genes and chromokinesins in the MCF10A cells uncovered established and previously unrecognized phenotypes, including nuclear morphology changes and reciprocal sign epistasis in DNA damage.

Staphylococcus haemolyticus is a common commensal bacterium but also an opportunistic pathogen, frequently implicated in bacteraemia and sepsis in preterm neonates and immunocompromised patients. Despite its clinical relevance, relatively little is known about the population structure of S. haemolyticus and how this relates to its ability to colonise humans or cause disease. In this study, we analysed commensal and clinical strains isolated from neonates and adults from 20 countries between 1957 - 2022. Whole genome sequencing of these isolates, combined with publicly available data, generated a comprehensive dataset of 986 genomes. This enabled us to characterise the species population structure and track the distribution of antimicrobial resistance (AMR) and virulence determinants. Our analysis revealed a highly diverse genome structure, with multiple phylogenetic groups showing distinct associations with commensalism or pathogenicity. We observed extensive variation in mobile genetic elements, prophages, and AMR genes, alongside increased carriage of plasmids and AMR genes over time. Furthermore, genes lined to metal homeostasis, detoxification, and oxidative stress tolerance were differently abundant between commensal and clinical isolates. This work provides the most detailed view to date of S. haemolyticus diversity, its evolutionary dynamics, and the genetic factors that may underpin the transition from commensal to pathogen.

ABSTRACT The global rise of convergent carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) represents a major clinical challenge, yet the role of virulence plasmids (pVirs) in shaping bacterial physiology and pathogenicity remains incompletely understood. Using a CRISPR-Cas9–based curing system, we precisely eliminated pVirs from two clinical CR-hvKp strains with distinct genetic backgrounds, ST23-KL1 ( bla NDM-1 ) and ST11-KL64 ( bla KPC-2 ). Loss of pVir conferred fitness advantages in vitro, reduced capsule production and hypermucoviscosity, and promoted biofilm formation, while markedly attenuating virulence in murine sepsis models. Despite this reduction, the pVir-cured ST23-KL1 strain retained higher virulence than the pVir-cured ST11-KL64 strain, underscoring the contribution of chromosomal background to pathogenic potential. Transcriptomic profiling revealed both shared and strain-specific transcriptional responses to pVir deletion, with broader perturbations observed in the ST11-KL64 strain. pVir removal had limited effects on antibiotic MICs. Complementation experiments further demonstrated differential regulatory roles of the rmpADC and rmpA2D2 operons in capsule expression and hypermucoviscosity across the two strains. Together, these findings establish pVirs as central determinants of CR-hvKp virulence and highlight complex host–plasmid interactions that influence bacterial adaptation and pathogenicity. IMPORTANCE The emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) poses a critical threat to global health, yet the contribution of virulence plasmids (pVirs) to bacterial fitness and pathogenicity remains poorly defined. By employing a CRISPR-Cas9–based curing strategy, we dissected the role of pVirs in two genetically distinct CR-hvKp strains and uncovered their multifaceted impact on capsule production, hypermucoviscosity, biofilm formation, and virulence. Our findings reveal that pVir loss confers fitness advantages in vitro while attenuating virulence in vivo, with strain-specific transcriptional responses and differential regulation by rmp operons. These results underscore the complex interplay between plasmid-encoded and chromosomal determinants in shaping CR-hvKp pathogenicity and adaptation, offering mechanistic insights that may inform future therapeutic strategies targeting plasmid-mediated virulence.

Triple pistil (TP) wheat is a historical genetic resource capable of producing up to three grains in a single floret and bolstering the current stagnant grain yield potential. TP phenotype is speculated to be the result of a spontaneous mutation; however, the exact underling genetic mechanism remains elusive, with lack of functional markers for early generation trait selection in hybrid wheat breeding programs. Here, scanning electron microscopy highlighted clear developmental differences between single and triple pistil plants started to arise during 1-2 cm long young spike stages. Using a forward genetics approach, we identified consistent TP-associated mutations in two genes ( TraesCS2D02G490900 and TraesCS2D02G491600 ) exhibiting a nearly complete co-segregation with TP phenotype and developed functional markers for early generation trait selection. CRISPR-Cas9 mediated gene-editing of TraesCS2D02G490900 shifted grain set toward single-grain florets in one edited line in transgenic wheat plants. Furthermore, grain yield evaluation exhibited a significant increase in grains per spike, with no statistically significant reduction in grain weight per spike. Hybrids between common and TP wheat exhibited relatively higher yields, highlighting TP wheat as a significant donor to fortify grain yield potential. This study provides co-dominant functional markers for early generation TP trait selection and valuable targets for hybrid wheat breeding programs.

ABSTRACT Tissues exhibit metabolic heterogeneity that tailors metabolism to their physiological demands. How the conserved pathways of metabolism achieve metabolic heterogeneity is not well understood, particularly in vivo. We established a system in Caenorhabditis elegans to investigate tissue-specific requirements for glucose 6-phosphate isomerase (GPI-1), a conserved glycolytic enzyme that also regulates the pentose phosphate pathway (PPP). Using CRISPR-Cas9 genome editing, we found that gpi-1 knockout animals display germline defects consistent with impaired PPP, and somatic defects consistent with impaired glycolysis. We discovered that two GPI-1 isoforms are differentially expressed and localized: GPI-1A is expressed in most tissues, where it displays cytosolic localization, whereas GPI-1B is primarily expressed in the germline, where it localizes to subcellular foci near the endoplasmic reticulum. GPI-1B expression alone is sufficient to maintain wild type levels of reproductive fitness, but insufficient to reconstitute wild-type glycolytic dynamics. Our findings uncover isoform-specific, spatially-compartmentalized functions of GPI-1 that underpin tissue-specific anabolic and catabolic metabolism in vivo , underscoring roles for subcellular localization in achieving tissue-specific metabolic flux.

Source–sink interactions play a critical but mechanistically underexplored role in coordinating reproductive output and longevity in plants. Here, we investigated the role of FT1 , the barley homolog of the florigen FLOWERING LOCUS T ( FT ), in regulating source (leaf) and sink (inflorescence) development and metabolism. Using ft1 knock-out mutants in the spring barley cultivar Golden Promise, which carries a mutated ppd-H1 allele, and in an introgression line with a wild-type Ppd-H1 allele, we show that Ppd-H1 primarily regulates the timing of inflorescence development and flowering through FT1 , whereas variation in tiller number and leaf size is determined by the genetic background. ft1 mutants exhibited reduced determinacy of both leaf and inflorescence meristems, resulting in increased leaf and spikelet numbers and size, but severely reduced inflorescence fertility, altered senescence patterns, and significantly extended plant longevity. The ft1 mutants exhibited a strong transcriptional reprogramming of genes involved in both the light and dark reactions of photosynthesis in the leaf, alongside an upregulation of genes associated with carbon catabolism and stress responses in the leaf and inflorescence. Elevated soluble sugar and starch levels in ft1 inflorescences indicated that the impaired development and fertility of ft1 inflorescences were not caused by carbon limitation, but instead reflected a reduced sink strength. Our work reveals that FT1 coordinates the development of vegetative and reproductive meristems and organs with plant physiology and metabolism, thereby regulating source–sink relationships and balancing plant longevity with reproductive output.

Hair cells of the zebrafish lateral line have proven to be a good model for studying hair cell function in a system that is easily genetically manipulated, rapidly develops and is experimentally accessible. However, characterization of potential developmental changes, and possible differences along lateral line position are lacking. Here, we used in vivo patch clamp to investigate the electrophysiological and exocytic properties of neuromast hair cells over early development across body location. Long depolarizations led to steady increases in membrane capacitance, presumably due to exocytosis of vesicles localized to ribbon synapses. The magnitude and kinetics of capacitance changes did not vary significantly across the L1 to L6 position of neuromasts along lateral line, but the magnitudes were found to be significantly smaller in hair cells found in the tail region across all developmental time points. For each region, we found no significant changes in capacitance responses between 3 and 7 days after fertilization. Hair cell capacitance responses were greatly reduced in animals injected with CRISPR/Cas9 with gRNAs targeted to otoferlin b. These results confirm the essential role of otoferlin b in neuromast hair cell function, and they establish the fidelity of CRISPR/Cas9 to rapidly mediate genetic removal of critical genes to study their impact on synaptic release.

ABSTRACT Gene editing, especially the CRISPR/Cas9 ( C lustered R egulatory I nterspaced S hort P alindromic R epeats/ C RISPR as sociated protein 9 ) system, has revolutionized trait development in crops. However, large parts of the world are missing out on applying CRISPR in planta . There is an obvious lack of gene editing applications in locally relevant crops in the Global South which tend to be neglected by mainstream agricultural research and development. Access barriers to these new breeding technologies need to be removed to allow the potential impact of these technologies on food security to happen. Here, we present the ENABLE® Gene Editing in planta toolkit, a minimal molecular toolbox allowing users to create a CRISPR knockout vector for transient or stable plant transformation in two simple cloning steps. We validate the toolkit in rice ( Oryza sativa ) protoplasts and in Arabidopsis thaliana plants. The ENABLE® kit is designed to be utilized specifically by users in the Global South who are new to CRISPR technology by providing a simple workflow, extensive accompanying protocols as well as options for low cost methods for cloning verification and gene editing verification in planta . We hope that our toolkit helps bridge the gap between the recent biotechnological advancements in plant breeding that high income countries can access and the lack of those technologies in low and middle income countries.

Understanding how individual genetic backgrounds shape the effects of disease-associated mutations is central to elucidating the biology of complex psychiatric disorders. We developed a scalable ‘village editing’ strategy that enables simultaneous genome editing across multiple induced pluripotent stem cell (iPSC) lines, allowing systematic assessment of how polygenic context modulates the impact of specific mutations. Using pooled CRISPR editing in 15 iPSC lines spanning a range of schizophrenia (SCZ) polygenic risk scores, we generated homozygous and heterozygous knockouts in two known SCZ-associated genes: LRP1 , involved in cholesterol import, and NRXN1 , a presynaptic adhesion molecule. By mixing all lines prior to editing and de-multiplexing them afterward, we efficiently produced multi-donor knockout neurons at scale. Transcriptomic profiling revealed that LRP1 and NRXN1 loss produce both shared and donor-specific effects on neuronal gene expression, with variable perturbation of neurotransmitter transport and cholesterol biosynthesis pathways across genetic backgrounds. These results demonstrate that village editing enables systematic dissection of gene-background interactions in human neurons, offering a powerful framework for studying the polygenic architecture of psychiatric disease.

The ULK1 complex (ULK1C) and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) act together to initiate autophagy. Human ULK1C consists of ULK1 itself, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P generated by PI3KC3-C1 is essential to recruit and stabilize ULK1C on membranes for ULK1 to phosphorylate its membrane-associated substrates in autophagy induction, even though ULK1C subunits do not contain any PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a tight complex with the PI3P-binding protein WIPI3, as well as with WIPI2. Bound to WIPI2-3, ATG13:ATG101 aligns with the membrane to insert its Trp-Phe (WF) finger into the membrane. Molecular dynamics simulations show that alignment of WIPIs and the ATG101 WF finger cooperatively stabilizes the complex on membranes, explaining the essential role of the WF residues in autophagy. Biochemical reconstitution and a cell-based assay show that WIPI3:ATG13 engagement is required for ATG16L1 phosphorylation by ULK1, ATG13 puncta formation, and bulk autophagic flux. We further showed that a kinase domain (KD)-proximal PVP motif within the ULK1 IDR docks onto the surface of the ATG13:ATG101 HORMA dimer and used molecular modeling to show how the ULK1 KD is brought close to the membrane surface. Biochemical reconstitution and cell-based assays show that the PVP motif is essential for in vitro ULK1 phosphorylation of ATG16L1 and important for BNIP3/NIX-dependent mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface downstream of PI3KC3-C1.

ABSTRACT Leishmania donovani (Ld), the etiological agent of visceral leishmaniasis, poses a significant global health burden due to its complex dixenous lifecycle involving both insect vectors and mammalian hosts. Successful infection in mammals requires the coordinated activity of stage-specific virulence factors. The zinc metalloprotease glycoprotease 63 (GP63) is a well-established virulence determinant critical for host cell attachment and invasion by insect-stage promastigotes. For humans, subsequent parasite propagation depends exclusively on intracellular amastigotes arising from lysed macrophages. Classical GP63 expression and function in Ld amastigotes remain poorly understood, and GP63 null mutants reportedly retain infectivity in mice, raising fundamental questions about virulence factor complementation during mammalian infection. By employing comparative transcriptomics, CRISPR-based mutagenesis, complemented with cell biology, and biochemical assays, this work identifies and characterizes multiple Ld GP63 paralogues with distinct roles in mammalian infection. While both copies of GP63 encoded on chromosome 10 (LdGP63_10.51 and 10.52) were functionally redundant, LdGP63_28 encoded on chromosome 28 proved essential for intracellular amastigote survival by suppressing host cell pyroptosis. Moreover, LdGP63_31 (chromosome 31) was found to primarily mediate promastigote attachment to the host macrophages with minimal contribution from LdGP63_28, facilitating initial infection establishment and amastigote genesis. Importantly, the absence of LdGP63_28 impacted amastigote infection more severely as compared to LdGP63_31. Structural and enzymatic analyses revealed divergent localization and substrate specificities to fulfil functional requirement of these divergent proteases, which have evolved independently to carry out diverse function in establishing infection. Collectively, this study indicates evolutionary divergence and functional specialization among GP63 isoforms in Ld by demonstrating that amastigote-specific and promastigote-specific GP63 isoforms synergistically mediate infection establishment and persistence.

ABSTRACT N 6 -methyladenosine (m 6 A) is the most prevalent internal modification of cellular and viral RNA and is critical to the regulation of its localization, stability, and translation. Previous studies on the role of m 6 A during HIV-1 replication have produced conflicting results. Since m 6 A function can vary dramatically by cell type and state, here we aimed to clarify the role of the m 6 A machinery during HIV-1 replication in primary CD4+ T cells. Using CRISPR-Cas9 we targeted 46 cellular genes implicated in m 6 A or 5-methylcytosine (m 5 C) regulation and measured subsequent HIV-1 replication in primary CD4+ T cells. Only knockout of the m 6 A writer complex auxiliary proteins VIRMA and WTAP, and the m 6 A reader YTHDF2 were validated as significantly decreasing HIV-1 replication. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in only marginal changes in HIV-1 infection, despite significant decreases in total cellular m 6 A levels. Chemical inhibition of METTL3 led to a dose-dependent decrease in HIV-1 infection, coupled with an increase in protein levels of METTL3 and other writer complex members. Expression of writer proteins was also co-dependent, revealing complex regulatory feedback mechanisms. Overall, these results clarify the role of epitranscriptomic machinery during HIV-1 replication in primary CD4+ T cells and suggest regulation by auxiliary members of the m 6 A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells. IMPORTANCE m 6 A is the most common chemical modification on cellular and viral RNA and regulates its stability, localization, and translation. m 6 A modification and its regulation varies dramatically between cell types and cell states. In this study, we investigated the role of m 6 A factors during HIV-1 infection of physiologically relevant primary CD4+ T cells. Using CRISPR-Cas9 to knockout 46 cellular genes implicated in RNA modification, we found only the m 6 A writer complex auxiliary members WTAP and VIRMA, and the reader YTHDF2, significantly affected HIV-1 replication in these cells. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in marginal changes in HIV-1 infection, despite larger reductions in total cellular m 6 A levels. Our findings suggest regulation by auxiliary members of the m 6 A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells.

Summary Phosphorus (P) is a vital macronutrient essential for plant growth, and its deficiency significantly hampers agricultural productivity. The PHOSPHATE 1 (PHO1) protein family, characterized by an N-terminal SPX domain, four transmembrane (4TM) domains, and a C-terminal EXS domain, is pivotal in transporting phosphate (Pi) from roots to shoots. Rice, PHO1;2 plays a crucial role in the Pi export process, and defects in this gene cause severe growth retardation and Pi deficiency symptoms even when external Pi levels are adequate. This study examined the roles of the EXS domain and the combined 4TM+EXS domains of OsPHO1;2 in supporting plant growth responses, independent of Pi transport activity, as well as their influence on hormone signaling and gene regulation. Using CRISPR/Cas9, rice lines expressing specific OsPHO1;2 domains (EXS or 4TM+EXS) were created by targeted deletion of particular domain-coding regions. Phenotypic analysis under Pi-sufficient and deficient conditions, as well as phosphate profiling, revealed that EXS lines exhibited notably better growth than loss-of-function mutants, ospho1;2, during early development, despite having similar shoot Pi levels to the null mutant. These lines showed lower levels of defense hormones (jasmonic acid) than ospho1;2 but were comparable to those in the wild type. Conversely, 4TM+EXS lines exhibited growth patterns similar to ospho1;2 mutants. RNA sequencing indicated that the phosphate starvation response (PSR) and defense pathways were less pronounced in the EXS lines compared to ospho1;2 mutants. However, both EXS and 4TM+EXS lines showed seed development defects and reduced total phosphorus content in seeds, mirroring the ospho1;2 phenotype. Heterozygous plants carrying one functional OsPHO1;2 allele displayed normal growth and seed development, indicating the mutation’s recessive nature. The findings suggest that the EXS domain of OsPHO1;2 can promote plant growth independently of Pi transport by decreasing PSR and modulating defense hormone pathways. This further suggests a signaling role for PHO1 domains beyond direct Pi translocation. Overall, these results enhance our understanding of Pi homeostasis and may help form strategies for breeding P-efficient crops.

Neuronopathic Gaucher disease (nGD) is a lysosomal storage disorder caused by GBA1 mutations, leading to defective acid β-glucosidase (GCase) and accumulation of glycosphingolipid substrates, causing inflammation and neurodegeneration. Patients with nGD manifests severe neurological symptoms, but current animal models fail to fully recapitulate human condition, posing a major barrier to the development of effective therapies targeting the brain. To bridge this gap, we have developed midbrain-like organoids (MLOs) from human induced pluripotent stem cells (hiPSCs) of nGD patients with GBA1 L444P/P415R and GBA1 L444P/RecNcil mutations to model nGD brain pathogenesis. These nGD MLOs exhibited GCase deficiency, resulting in diminished enzymatic function, accumulation of lipid substrates, widespread transcriptomic changes, and impaired dopaminergic neuron differentiation, mirroring nGD pathology. GBA1 mutation correction mediated by CRISPR/Cas9 restored GCase activity, normalized lipid substrate levels, and rescued dopaminergic neuron function, confirming the causal role of GBA1 mutations during early brain development. Using this novel platform, we further evaluated therapeutic strategies, including SapC-DOPS nanovesicles delivering GCase, AAV9-GBA1 gene therapy, and substrate reduction therapy with GZ452, a glucosylceramide synthase inhibitor currently under clinical investigation. These treatments either restored GCase activity, reduced lipid substrate accumulation, improved autophagic and lysosomal abnormalities, or ameliorated dysregulated genes involved in neural development. These patient-specific, 3D neural models offer a transformative, physiologically relevant platform for unravelling disease mechanisms and accelerating the discovery of therapies for patients with nGD.

ABSTRACT The ryanodine receptor (RYR) genes encode evolutionarily conserved calcium release channels involved in a wide range of calcium-dependent biological processes. Here we show that the sole Drosophila RYR gene ( dRyR ) functions in differentiated somatic and cardiac muscle as well as in developing embryonic myotubes. In the larval body wall muscles, dRyR protein localizes at the SR membranes and dRyR knockdown adversely affects muscle contractility, suggesting its conserved role in calcium-triggered E-C coupling. After dRyR attenuation, sarcomere and mitochondrial patterns are severely impaired, showing dRyR involvement in structural muscle properties. However, dRyR is also prominently expressed and functionally required in growing embryonic muscles. dRyR loss of function leads to myotube growth defects and thin myofiber phenotypes, while its overexpression induces myofiber splitting. Given the structural and functional conservation of dRyR , we used Drosophila to test the impact of one human RYR1 variant of unknown significance (VUS). Larvae carrying p.Met4881Ile RYR1 VUS showed impaired mobility and altered structural muscle properties reminiscent of those seen in dRyR knockdown, thus indicating it is likely pathogenic. Overall, we show that Drosophila dRyR plays a conserved role in setting muscle contractility and structural muscle features. Our findings underline the still under-investigated role of dRyR as a promyogenic factor and provide a first example of the impact assessment of a human RYR1 VUS in Drosophila .

Prion diseases are transmissible neurodegenerative disorders caused by the spread of misfolded prion protein between cells and individuals, yet the paths by which prions colonize cells are undefined. Here we map the determinants of prion uptake with a genome-wide quadruple-guide CRISPR activation screen. Uptake of prions was measured by flow cytometry in PRNP -ablated human SHSY-5Y cells exposed to synthetic ovine prions. We identified 43 genes modulating prion uptake, 6 of which belonged to the core components of the Bone Morphogenetic Protein (BMP) signaling axis. Prion internalization was increased by overexpression of BMP receptors (BMPR1B, BMPR2, ACVRL1) and the Small Mothers Against Decapentaplegic (SMAD1 and SMAD5) intracellular BMP effectors, whereas the inhibitory SMAD6 reduced it. The internalization of other proteopathic seeds (α-synuclein, tau K18, amyloid-β) and other endocytic probes (transferrin, dextran, E. coli bioparticles) was unaffected. Transcriptional profiles of a panel of persistently prion-infected cell lines showed broad dysregulation of BMP-related signaling. Hence, BMP signaling is a gatekeeper of prion entry mechanistically distinct from bulk endocytosis and amyloid uptake. Chronic prion propagation induced SMAD1/5 phosphorylation, suggesting a positive-feedback loop during prion infection. The connection of prion uptake to a developmentally conserved morphogen pathway with rich pharmacology suggests that BMP signaling nodes may serve as tractable levers to modulate early events in prion transmission.

The Maternal Embryonic Leucine Zipper Kinase (MELK) gene is a part of the Snf1/AMPK of serine/threonine kinase family. MELK has recently attracted considerable interest in the fields of stem cell and cancer biology. Furthermore, MELK is expressed normally during embryogenesis and in proliferative tissues; however, its aberrant overexpression has been observed in various malignancies, including glioma, breast, lung, colorectal, gastric, and hematological cancers. Higher MELK levels are often correlated with unfavorable prognosis, aggressive tumor manifestations, resistance to treatment, and stem-like tumor morphologies. Preclinical studies utilizing RNA interference and small-molecule inhibitors such as OTSSP167 demonstrate that MELK promotes cancer cell proliferation, survival, and metastasis. However, contrasting evidence from CRISPR/Cas9-based knockout studies indicates that MELK may not be essential for tumor growth, raising concerns that the observed anti-tumor effects of MELK inhibitors could partly result from off-target activity. This review aims to summarize the current understanding of MELK biology, including its functions in cell cycle regulation, apoptosis, oncogenic signaling pathways, and tumor stemness. In this review, we discuss the therapeutic potential and limitations of MELK inhibitors, the controversy regarding MELK dependency, and the implications for cancer diagnosis and treatment. MELK may not be a universal driver oncogene; nonetheless, it is consistently linked to aggressive disease, underscoring its potential as a prognostic biomarker and a candidate for therapeutic co-targeting in combination treatments.

Human norovirus (HNoV) is a leading cause of acute gastroenteritis, yet the mechanisms by which it interfaces with host innate immunity remain elusive. Here, we demonstrate that the HNoV NS7 protein, an RNA-dependent RNA polymerase, acts as a direct activator of canonical inflammasomes. Using reconstituted cell systems and human intestinal enteroids (HIEs), we found that NS7 interacts with both NLRP3 and NLRP6, promoting ASC speck formation, caspase-1 cleavage, and secretion of IL-1β and IL-18. HNoV infection of HIEs recapitulated these events, including gasdermin D processing and robust IL-18 release. Importantly, CRISPR/Cas9-mediated NLRP6 deficiency abrogated inflammasome activation and markedly enhanced viral replication, underscoring the essential role of NLRP6 in epithelial antiviral defense. These findings identify NS7 as a novel inflammasome activator and establish NLRP6 as a key determinant of innate immune control of HNoV. Our study highlights inflammasome signaling as a potential therapeutic target for norovirus infection. Author summary In this study, we used human intestinal organoid models to explore how norovirus infection triggers a specific immune response known as inflammasome, which helps protect the gut from viral invaders. We focused on a viral protein called NS7 and discovered that it directly activates two types of inflammasome sensors, NLRP3 and NLRP6. We found that NLRP6, which is abundant in the gut lining, is especially important for detecting norovirus and launching an immune response. When we removed NLRP6 from intestinal cells, the virus was able to replicate more easily, and normal immune activation was lost. Our results reveal that norovirus uses its NS7 protein to interact with the body’s immune machinery in the intestine, and that NLRP6 plays a key role in controlling infection. This work highlights a new way in which the gut senses and responds to norovirus and may help guide future efforts to develop treatments that target these immune pathways.

ABSTRACT Serum antibodies from prior immune responses regulate B cell activation and germinal center (GC) access upon recall immunization. However, how antibodies produced by an ongoing immune response influence the outcomes of contemporaneous GCs is less clear. To explore this, we developed mouse models enabling the targeted ablation of plasma cells and antibodies produced by an immune response of interest, without affecting those produced homeostatically or by prior antigen encounters. Our findings show that, whereas antibody-mediated feedback is not required for affinity maturation, it can influence competition between B cells with different epitope specificities, specifically by reducing the abundance of clones that recognize the same epitopes as circulating antibodies. This modality of feedback represents a mechanism by which antibody responses can influence epitope specificity in ongoing GCs. These findings may therefore have implications for vaccination strategies aimed at steering clonal selection towards desired epitopes on complex antigens.

Eukaryotic flagella, or motile cilia, are iconic molecular machines whose beating drives cell propulsion and fluid transport across diverse organisms. Beat type and waveform are tailored to function, differing between species and cell types, and individual flagella can switch between beat types. Aberrant beating causes ciliopathies and infertility in humans 1 and prevents unicellular parasite transmission 2 . Eight distinct dynein motor protein complexes bind to axonemal doublet microtubules (DMTs) within flagella and drive beating, yet despite extensive structural analysis 3–5 , how this machinery achieves different beat types is unknown. Here, using the flagellate unicellular parasite Leishmania , we show a division of labour where specific dyneins drive specific beat types. Using cryo-EM, we determined the structure of the 96-nm repeat unit of the DMT and identified its dynein composition. We used CRISPR–Cas9 to systematically delete all 96-nm repeat proteins, comprehensively mapping necessity for swimming, and determined the contribution of each dynein to incidence and waveform of the preferred beat types. Outer dynein arms (ODAs) were required for symmetric tip-to-base beats, specific single-headed inner dynein arms (IDAs) were important for asymmetric base-to-tip beats (IDA d ), and double-headed IDA f important for both. This systematic analysis indicates that the prevailing dogma that ODAs drive and IDAs shape the beat 6–9 is either incomplete or not universal, and establishes new hypotheses for how different species, cell types and individual flagella achieve their necessary beat types.

Abstract In breast cancer, nuclear localization of Kaiso (ZBTB33), a dual specificity transcription factor, is a hallmark of high grade ductal-type carcinomas, especially in estrogen receptor negative disease. Regulation of gene expression by Kaiso is orchestrated via its engagement to distinct Kaiso binding sequence (KBS) motifs defined as canonical (cKBS; TCCTGCNA ) or CG-containing palindromic KBS (CG-KBS; TCTCGCGAGA ), depending on context. While there exists a clinical connection between localization of Kaiso expression and breast cancer, it remains unclear how Kaiso controls bi-modal canonical versus noncanonical transcriptional modulation. Here, we have combined Kaiso-specific chromatin immunoprecipitation (ChIP) and Kaiso-Dam methyltransferase identification (Dam-ID) approaches to map genomic Kaiso binding sites in breast cancer cells. We find that Kaiso mostly occupies the non-canonical CG-KBS sites in transcriptionally active and hypo-methylated (H3K4me3 and H3K27Ac enriched) promoter regions. Noncanonical CG-KBS targets are actively transcribed and linked to fast biological processes such as metabolism, cell cycle regulation and DNA damage repair. Functionally, we show that Kaiso expression is essential to prevent DNA damage in breast cancer cells. Loss of Kaiso leads to reduced expression of DNA damage response gene expression and treatment with the chemotherapeutic agent cisplatin leads to overt accumulation of DNA damage in cells devoid of Kaiso. Our data thus favor a model in which Kaiso promotes fast transcriptional activation of key cellular processes in cancer cells through binding of its non-canonical consensus site.

Abstract Background Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta-coronavirus (PDCoV), and porcine rotavirus-A (PoRV) G9 are major swine pathogens primarily responsible for gastrointestinal diseases, particularly affecting lactating piglets and resulting in significant economic losses, especially in China. This study introduces a novel CRISPR-based nucleic acid detection method that integrates the high specificity of huLbCas12a with the sensitivity of loop-mediated isothermal amplification (LAMP) technology. Central to this method, the crRNA/Cas12a complex, with a molecular weight of approximately 144 kDa, enhances diagnostic accuracy through targeted gene editing. Incorporating fluorescence report probes and a lateral flow dipstick assay, this approach establishes a visual detection system capable of simultaneously identifying all four viruses. Results It enables the visualization of viral genomes from as low as 1 to 10 copies/µL without cross-reactivity. In comparative testing of 95 clinical samples, our quadruplex LAMP-CRISPR assay demonstrated 100% concordance with RT-qPCR for the three porcine coronaviruses and 98.94% concordance with RT-qPCR for PoRV G9. Conclusions Offering a robust and reliable tool for on-site virus detection, this method significantly aids in the timely prevention of virus spread and mitigates its impact on the pig farming industry, demonstrating its critical role in enhancing biosecurity and disease management in veterinary contexts.

Damage to the vascular endothelium is a major contributor to acute radiation injury in multiple organs that underlies acute radiation syndrome (ARS), yet there are no FDA-approved radiation countermeasures targeting endothelial cells. Use of kinome-scale CRISPR screens performed in cultured human vascular endothelial cells isolated from different organs identified CLK2 as a potential radioprotective target. Pharmacological inhibition of CLK2 using TG003 and Cirtuvivint protected these endothelial cells against radiation injury and reversed its effects across the transcriptome and phospho-proteome. Human Organ Chip models of human intestine and lung that contain organ-specific epithelium and microvascular endothelium faithfully replicated clinical features of ARS when exposed to radiation, which were prevented when treated with CLK2 inhibitors. Thus, CLK2 inhibitors may represent a new class of radiation countermeasure drugs that can protect multiple organs against radiation-induced toxicities in patients with ARS.

Apolipoprotein E ( APOE ) genotype is well known to influence both amyloid-β (Aβ) and tau pathologies and risk for Alzheimer’s disease (AD), but it also affects α-synuclein (α-syn) levels, Lewy pathology and risk of dementia in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). The APOE-R136S (Christchurch, CC) point mutation has been shown to protect against AD pathology and dementia, however, the molecular mechanisms underlying this protection and its effects on α-syn pathology are not well understood. Using CRISPR/Cas9 technology, we created a CC arginine-to-serine point mutation at the conserved location in mouse APOE (R128S) to understand its effects on Aβ, tau and α-syn pathologies. We crossed these APOE CC mice to 5xFAD, PS19 and A53T-αSyn-GFP (A53T) mice. Using these various double mutant mice, we tested the effect of mouse APOE CC on different proteinopathies, including Aβ, tau, Aβ-induced tau after paired helical filament (PHF)-tau intracortical injections, and α-syn after preformed fibril (PFF) intracortical and intramuscular injections. We used immunohistochemical, biochemical and behavioral measures to test for protective effects of APOE CC on these different proteinopathies. Heterozygous (Het) and homozygous (Hom) APOE CC mice showed increased plasma cholesterol and triglyceride levels, as seen in humans, but no differences in body or brain weight, or life expectancy. APOE CC decreased Aβ-induced tau pathologies in PHF-tau injected 5xFAD;Hom mice but did not change Aβ-plaque pathology in 5xFAD mice or tau pathology in PS19 mice. Although Aβ levels, tau levels and mouse sex correlated strongly with the behavioral performance, we only detected subtle effects of APOE CC on anxiety-like behaviors in crosses with 5xFAD, PS19 and PHF-tau injected 5xFAD mice. Interestingly, Het and Hom APOE CC mice both showed reduced formation and spread of Lewy pathology in brain after intracortical α-syn PFF injection and reduced formation in spinal cord after α-syn PFF injection into the hindlimb gastrocnemius muscle in A53T mice. Our study emphasizes the protective effects of the APOE CC variant against different proteinopathies important for dementia and movement disorders, including Aβ plaque, tau and α-syn, and suggests that targeting APOE CC could provide new therapeutic strategies for AD, DLB and PD. Graphical Abstract

Objective Primary liver cancer is a leading cause of cancer-related mortality and harbors recurrent mutations in chromatin regulators such as BRCA1-associated protein 1 (BAP1), yet their functional impact remains unclear. We investigated how BAP1 deficiency affects liver homeostasis and tumorigenesis to clarify its functional role. Design We employed inducible, liver-specific BAP1 knockdown in mice subjected to diet-induced metabolic stress (including rescue experiments), alongside autochthonous hydrodynamic CRISPR models, and profiled livers by RNA-seq, immunohistochemistry, and mass spectrometry-based lipidomics. Complementary mechanistic assays in liver cancer cells examined the unfolded protein response (UPR) under endoplasmic reticulum (ER) stress; findings were supported by immunohistochemical and transcriptomic analyses of BAP1-mutant patient samples. Results BAP1 safeguards liver homeostasis under diet-induced metabolic stress, as its loss triggers ER stress, hepatocyte death, and acute liver failure. Lipidomics revealed a shift toward ER-stress-associated dyslipidemia, and transcriptomics showed negative enrichment of fatty-acid metabolism and positive enrichment of UPR pathways. In contrast, BAP1 loss synergizes with oncogenic drivers to accelerate tumorigenesis in autochthonous liver cancer models, underscoring its context-dependent tumor suppressor function. Mechanistically, BAP1 directly regulates the ER stress mediator DDIT3 (CHOP) through chromatin remodeling, linking BAP1 loss to maladaptive stress responses. Consistently, elevated CHOP expression was observed in BAP1-mutant human liver cancers and other tumor types. Conclusion These findings establish BAP1 as a key chromatin regulator that connects stress adaptation to both liver homeostasis and tumorigenesis, highlighting the BAP1-UPR axis for future translational assessment. What is already known on this topic? BAP1 is a recurrently mutated chromatin regulator across cancers, including primary liver cancer, but its functional role in liver biology and tumorigenesis has remained unclear. Metabolic dysfunction-associated liver disease is a growing driver of liver tumorigenesis and is characterized by lipid imbalance, ER stress, and activation of the unfolded protein response. What this study adds? We show that BAP1 is a key chromatin regulator that integrates metabolic and ER stress responses in the liver. Loss of BAP1 undermines cellular adaptation to metabolic challenge and cooperates with oncogenic signals to promote liver tumorigenesis via dysregulated DDIT3 (CHOP) expression, linking chromatin control to hepatic stress resilience and disease progression. How this study might affect research, practice or policy? Our findings position the UPR-CHOP axis as a candidate therapeutic vulnerability in BAP1-deficient liver cancers, particularly in the context of MASLD/MASH, and provide a conceptual framework for targeting stress adaptation pathways in precision oncology.