Microcystins are potent cyanotoxins produced by toxigenic cyanobacteria during harmful algal blooms (HABs), posing risks to ecosystems and human health. In this study, we developed a portable RPA-CRISPR/Cas12a biosensing platform for the rapid, on-site detection of the microcystin synthetase E ( mcyE ) gene, a key biomarker for microcystin-producing strains. The developed RPA-CRISPR/Cas12a assays enable detection of the mcyE gene within 50 min, with either fluorescence or lateral flow assay readouts. The fluorescence readouts have an analytical detection limit of 48.4 copies/µL and a dynamic range of 1.2 × 10 2 to 1.2 × 10 7 copies/µL. To enable field deployment, a magnetic bead-based DNA extraction method was integrated, achieving extraction within 1 hour without centrifugation. The complete workflow demonstrated a method LOD of 8.4 × 10 2 cells/mL in spiked lake water. Applicability was validated using non-spiked environmental water samples collected from multiple HAB-affected lakes. Importantly, a systematic matrix effect assessment was conducted for the CRISPR sensing step, evaluating environmental variables such as pH, ions, nutrients, and natural organic matter. This study establishes a practical, sensitive, and selective detection tool for proactive HAB monitoring. The platform’s simplicity, portability, and completeness, from sample pretreatment to signal readout, highlight its potential for real-world environmental biosensing applications.

Prostate cancer is a major cause of cancer-related deaths among men in Sub-Saharan Africa, where late-stage diagnoses are common due to limited access to affordable and sensitive diagnostic tools. Early detection is essential to improve survival and reduce the disease burden. This review explores the integration of epigenetic biomarkers and CRISPR-Cas12a technology as a transformative approach for early, non-invasive prostate cancer detection in resource-limited settings. Among the many complexities of cancer development, molecular dysregulation plays a remarkable role, and epigenetic modifications such as DNA methylation, histone changes, and non-coding RNA expression have emerged as stable and specific biomarkers with significant potential for the early detection and characterisation of prostate carcinogenesis. However, their low concentration in body fluids presents a detection challenge. CRISPR-Cas12a, known for its high specificity and sensitivity, offers a promising solution. When combined with isothermal amplification and liquid biopsy techniques, it enables rapid, low-cost, and point-of-care diagnostics. This review proposes a low-cost, CRISPR-Cas12a-based diagnostic pipeline for detecting prostate cancer-specific epigenetic markers in liquid biopsies. The implementation of this technology in Sub-Saharan Africa could significantly improve early diagnosis, reduce mortality, and advance health equity.

Abstract Long-Read Sequencing (LRS) technologies offer capabilities for characterizing complex engineered DNA constructs from Golden Gate and barcoded DNA variant assemblies, CRISPR engineered libraries or Multiplexed Assays of Variant Effect (MAVE) experiments. However, the heterogeneity of such molecules, combined with potential structural and length variability, presents analytical challenges. We present SLICER (Sequencing Long-read Identifier of Complex Element Regions), a pipeline for analyzing LRS of such constructs. SLICER dynamically identifies and extracts user-defined barcode/core elements per-read using an anchor-based method, accommodating positional/length variations and aligns these back to reference sequences. If absent, SLICER is capable of de novo reference prediction, a feature that can be insightful to identify unpredicted/aberrant phasing/combinatorial events. When benchmarked, SLICER’s d e novo reference prediction was accurate to within 1% of reference data. SLICER’s dynamic extraction and robust de novo reference capabilities provide an invaluable tool for synthetic and engineered biology applications, enabling comprehensive interrogation of complex barcoded DNA constructs and libraries. SLICER is available at https://github.com/mbassalbioinformatics/SLICER.

Abstract Innate immunity, traditionally viewed as non-specific, is increasingly recognized for its capacity to regulate microbial communities with precision. In the sea anemone Nematostella vectensis , we uncover a form of selective immunity mediated by nematosomes—motile immune cell clusters that preferentially phagocytose foreign Vibrio isolates while sparing native bacteria. We identify the transcription factor cJUN as essential for this process: CRISPR/Cas9-mediated knockout of cJUN impairs nematosome proliferation, reduces lysosomal activation, and alters microbiome composition by allowing colonization of non-native strains. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit immune discrimination. Our findings challenge the classical dichotomy between innate and adaptive immunity and reveal that immune specificity may be evolutionarily ancient. This work establishes Nematostella as a model for studying microbiome-induced innate immune training and highlights conserved mechanisms that maintain host-microbe homeostasis.

ABSTRACT Understanding the dynamic regulation of signaling pathways requires methods that capture cellular responses in real time. While high-content imaging-based genetic screens have transformed functional genomics, they have remained largely limited to static or binary phenotypes. Here, we present DynaScreen, an imaging-based, pooled CRISPR screening platform that enables high-throughput investigation of dynamic cellular phenotypes at single-cell resolution. By integrating Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) biosensors with photoactivation-based single-cell tagging and pooled CRISPR screening technology, we establish a scalable system to identify genes that regulate the timing, amplitude, and duration of signaling responses. As proof of principle, we applied this approach to the cAMP signaling pathway, a key regulator of cellular physiology. Using a custom guide RNA (gRNA) library, we tracked real-time cAMP dynamics in response to agonist stimulation and identified genes that modulate its basal levels and response kinetics. Cells with aberrant signaling were selectively photoactivated, isolated by fluorescence-activated cell sorting (FACS), and subjected to next-generation sequencing to pinpoint causal genetic perturbations. This strategy successfully uncovered known and novel regulators of cAMP dynamics. In conclusion, the integration of FLIM microscopy, CRISPR technology and open-source software to handle image analysis, automated hit identification and data representation, enables real-time exploration of dynamic phenotypes in a wide range of biological settings.

Summary Mitochondria contain their own genome, the mitochondrial DNA (mtDNA), which is under strict control of the cell nucleus. mtDNA occurs in many copies in each cell, and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the cell- and tissue-specific impact of mtDNA mutations, ultimately giving rise to rare mitochondrial and common neurodegenerative diseases. However, little is known about how copy number and heteroplasmy interact within single cells, and how this is regulated by the nuclear genes and pathways that sense and control them. Here we describe MitoPerturb-Seq for CRISPR/Cas9-based high-throughput single-cell interrogation of the impact of nuclear gene perturbation on mtDNA copy number and heteroplasmy. We screened a panel of nuclear mtDNA maintenance genes in cells with heteroplasmic mtDNA mutations. This revealed both common and perturbation-specific aspects of the integrated stress-response to mtDNA depletion, that were only partially mediated by Atf4, and caused cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq thus provides novel experimental insight into disease-relevant mito-nuclear interactions, ultimately informing development of novel therapies targeting cell- and tissue-specific vulnerabilities to mitochondrial dysfunction.

The transcription factor (TF) GATA4 is a key mediator of cardiogenesis. GATA4 regulates cardiogenesis through the expression of its target genes, only some of which have been identified. We have used a gain of function model based on pluripotent embryonic ectoderm explants from Xenopus embryos expressing GATA4, to identify a set of downstream targets of GATA4 which are also regulated by Nodal, a known cardiogenic signal. GATA4 was shown to be required for the expression of target genes tbx2 and prdm1 in vivo, likely acting in a direct fashion by interacting with their regulatory regions. In addition, tbx2 and prdm1 are shown to have roles of their own in vivo, as downregulation of tbx2 , a positive target, and overexpression of prdm1 , a negative target, interferes with cardiac development in Xenopus embryos. The conservation of the GATA4-TBX2-PRDM1 regulatory relationship was shown in human iPSC-derived cardiomyocytes. Loss of function of GATA4 lead to downregulation of TBX2 , upregulation of PRDM1 expression and failure of cardiogenesis. GATA4-deficient cells failed to form normal cardiomyocytes, with most cells adopting alternative fates and only a small minority expressing an aberrant cardiomyocyte phenotype. Genome-wide transcriptomic analysis documented severe reduction of cardiomyocyte and endothelial cell transcriptomes and upregulation of transcriptional profiles of smooth muscle cells and fibroblasts. Disruption of TBX2 function did not alter cardiomyocyte differentiation efficiency but led to the formation of hypertrophic cardiomyocytes characterised by defective sarcomeres and deficient calcium signalling. In addition, we show that whilst PRDM1 is not essential for formation of cardiomyocytes it is implicated in suppression of alternative cell fates. The results presented establish a conserved regulatory relationship between GATA4 and its target genes TBX2 and PRDM1 and roles for these genes in the modulation of cardiomyocyte development, expanding the cardiac gene regulatory network and providing further insight into how cardiogenesis proceeds.

The MAST family of serine/threonine kinases has been implicated in a spectrum of human neurodevelopmental disorders. However, little is known about their biological function or regulation. Seeking to fill these gaps in our knowledge, we have identified upstream and downstream partners of MAST1. 14-3-3η, a neuronal 14-3-3 paralog, specifically interacts with MAST1 at two regulatory serines, S90 and S161. PAK, a neuronal regulator of the actin cytoskeleton, phosphorylates MAST1 to regulate its interaction with 14-3-3η. Exploiting mouse models of human Mega Corpus Callosum Syndrome (MCC) and whole brain phosphoproteomics, we identify the microtubule-associated protein Tau as a substrate of MAST1. We show that pathogenic MAST1 mutations perturb protein function either through misfolding or attenuation of kinase activity. Our data is consistent with a model in which the MAST kinases couple PAK, a neuronal regulator of the actin cytoskeleton, to microtubule remodeling during the differentiation and specification of cortical neurons.

Harnessing the precision of CRISPR systems for diagnostics has transformed nucleic acid detection. However, the integration of upstream cellular signals into CRISPR-based circuits remains largely underexplored. Here, we introduce a synthetic transduction platform that directly links endogenous DNA repair activity to CRISPR-Cas12a activation. By coupling base excision repair (BER) events to a programmable DNA-based transducer, our system converts the activity of DNA glycosylases, such as uracil-DNA glycosylase (UDG) and human 8-oxoguanine glycosylase (hOGG1), into a robust fluorescence signal via Cas12a-mediated trans-cleavage. This one-step CRISPR-based assay operates directly in cell lysates, enabling rapid and sensitive readout of enzymatic activity with high specificity. Additionally, it also enables in 15 minutes the throughput screening of novel potential inhibitors with high sensitivity. The modular design allows adaptation to diverse repair enzymes, offering a generalizable strategy for transforming intracellular repair events into programmable outputs. This approach lays the foundation for activity-based molecular diagnostics, synthetic gene circuits responsive to cellular states, and new tools for monitoring DNA repair in real time and drug screening.

Noncoding genetic variants underlie many complex diseases, yet identifying and interpreting their functional impacts remains challenging. Late-onset Alzheimer’s disease (LOAD), a polygenic neurodegenerative disorder, exemplifies this challenge. The disease is strongly associated with noncoding variation, including common variants enriched in microglial enhancers and rare variants that are hypothesized to influence neurodevelopment and synaptic plasticity. These variants often perturb regulatory sequences by disrupting transcription factor (TF) motifs or altering local TF interactions, thereby reshaping gene expression and chromatin accessibility. However, assessing their impact is complicated by the context-dependent functions of regulatory sequences, underscoring the need to systematically examine variant effects across diverse tissues, cell types, and cellular states. Here, we combined in vitro and in vivo massively parallel reporter assays (MPRAs) with interpretable machine-learning models to systematically characterize common and rare variants across myeloid and neural contexts. Parallel profiling of variants in four immune states in vitro and three mouse brain regions in vivo revealed that individual variants can differentially and even oppositely modulate regulatory function depending on cell-type and cell-state contexts. Common variants associated with LOAD tended to exert stronger effects in immune contexts, whereas rare variants showed more pronounced impacts in brain contexts. Interpretable sequence-to-function deep-learning models elucidated how genetic variation leads to cell-type-specific differences in regulatory activity, pinpointing both direct transcription-factor motif disruptions and subtler tuning of motif context. To probe the broader functional consequences of a locus prioritized by our reporter assays and models, we used CRISPR interference to silence an enhancer within the SEC63-OSTM1 locus that harbors four functional rare variants, revealing its gatekeeper role in inflammation and amyloidogenesis. These findings underscore the context-dependent nature of noncoding variant effects in LOAD and provide a generalizable framework for the mechanistic interpretation of risk alleles in complex diseases.

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 ( Plm Cas12e), a class II CRISPR system derived from Planctomycetes , a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Streptococcus pyogenes Cas9 ( Sp Cas9) and Staphylococcus aureus Cas9 ( Sa Cas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2 Max , for cleavage of CCR5 , a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural CCR5 Δ32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2 Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of CCR5 . Structural modeling indicated that two of the CasX2 Max substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2 Max in cellular gene excision. In sum, CasX2 Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.

The rapid advancement of genetic editing technologies, such as CRISPR-Cas9, has introduced unprecedented opportunities and challenges within professional sports. This study aims to systematically evaluate the legal and ethical implications associated with the application of gene editing among elite athletes. Employing a mixed-methods design, we conducted a comprehensive survey of 312 stakeholders-including athletes, coaches, legal experts, and ethicists-across five continents. Advanced statistical analyses, including Structural Equation Modeling (SEM) and Multivariate Logistic Regression, were utilized to identify significant predictors of legal risk perception and ethical concern. Results reveal a pronounced divergence in stakeholder attitudes: while 68% of legal professionals emphasize regulatory gaps, 74% of athletes express uncertainty regarding long-term health consequences. The SEM model demonstrated that perceived fairness (β=0.41, p<0.001) and regulatory clarity (β=0.36, p<0.001) are the strongest predictors of overall acceptance. These findings underscore the urgent need for robust international frameworks to address the multifaceted risks of gene editing in sports and highlight the importance of transparent policy-making. Our research provides actionable insights for regulators, sports organizations, and bioethics committees to anticipate and manage the evolving landscape of genetic technologies in athletics.

Leishmania amastigotes ingested by female phlebotomine sand flies are exposed to a harsh and dynamic environment, markedly different from that of their mammalian host. Within the sand fly’s alimentary tract, these parasite forms encounter shifts in temperature, pH and nutrient availability, which trigger significant morphological and physiological adaptations. Membrane transporter proteins, channels and pumps play a crucial role in facilitating the movement of solutes across eukaryotic membranes. Previously, a systematic loss-of-function screen of the L. mexicana “transportome” identified forty transporter deletion mutants that caused significant loss of fitness in macrophage and mouse infections. Here, using an independently generated library of over 300 barcoded gene deletion mutants, we monitored their growth fitness for seven days in vitro and tested which transporters are required for Leishmania promastigotes to successfully colonise Lutzomyia longipalpis sand flies for nine days. Overall, fitness scores correlated between promastigotes from long-term in vitro culture and in vivo sand fly infections. More importantly, for 34 mutants, a significant loss of fitness was observed exclusively in vivo . Moreover, deletion of the vacuolar H + ATPase (V-ATPase) proved detrimental for parasite persistence and promastigote differentiation in the sand fly, uncovering a key role for the V-ATPase at different stages throughout the Leishmania life cycle. Author Summary Leishmania parasites cause leishmaniases - a group of neglected tropical diseases that affect millions of people worldwide. These parasites must survive in two radically different environments: inside a mammalian host and within the gut of a blood-feeding sand fly. To thrive in the sand fly, Leishmania undergo extensive physiological changes and depend on transporter proteins to move nutrients and other molecules across their cell membranes. In this study, we focused on identifying which of these transporters are critical for the parasite’s survival inside the sand fly. We used a genetically engineered library of Leishmania promastigotes - the parasite form adapted to the insect vector - to assess the importance of more than 300 different transporter genes. We discovered that 34 of these transporters are essential for successful colonization of the sand fly. Among them, one key protein complex - the vacuolar H + ATPase (V-ATPase) pump – was found to be crucial for parasite survival in the insect vector. Our findings deepen our understanding of how Leishmania adapts to life within the sand fly and highlight potential molecular targets for disrupting its transmission.

Lung cancer histological subtypes include lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC). While typically distinct, combined LUAD/SCLC histology tumors occur, and LUAD can transform into SCLC as a resistance mechanism to targeted therapies, especially in EGFR -Mutant LUADs with RB1 / TP53 -inactivation. Although PRC2 complex expression increases during this transformation, its functional role has remained unclear. Using CRISPR-based autochthonous immunocompetent GEMMs, we demonstrate that inactivation of EED, the core PRC2 scaffolding subunit, impairs SCLC tumorigenesis and drives histological transformation from ASCL1-positive SCLC to LUAD through a transient NEUROD1-positive intermediate state. Mechanistically, EED loss de-represses bivalent genes co-marked by H3K27me3 and H3K4me3, including LUAD oncogenic RAS, PI3K, and MAPK pathway genes, to promote transformation to LUAD. Consistently, these same signaling genes are bivalently repressed in human SCLC patient-derived xenograft (PDX) tumors, suggesting a conserved PRC2-dependent mechanism to repress LUAD lineage oncogenic signaling to maintain the SCLC neuroendocrine identity. In a complementary EGFR -mutant LUAD GEMM with RB1/TP53 inactivation, EED was required for LUAD-to-SCLC transformation and distant metastasis upon EGFR withdrawal. These findings identify the PRC2 complex as a key epigenetic enforcer of SCLC neuroendocrine identity and nominate EED inhibition as a potential strategy to block SCLC transformation in high-risk LUAD.

Bacteria encode an enormous diversity of defense systems including restriction-modification and CRISPR-Cas that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate many recently identified anti-phage defense operons are comprised of a predicted nuclease and an accessory NTPase protein, suggesting additional classes of nucleic acid targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyze 16 nuclease-NTPase systems and define shared features that control anti-phage defense. Purification, biochemical characterization, and in vitro reconstitution of nucleic acid targeting for each system demonstrate protein–protein complex formation is a universal feature of nuclease-NTPase systems and explain patterns of phage targeting and susceptibility. We show that some nuclease-NTPase systems use highly degenerate recognition site preferences to enable exceptionally broad nucleic acid degradation. Our results uncover shared principles of anti-phage defense system function and provide a foundation to explain the widespread role of nuclease-NTPase systems in bacterial immunity.

Drosophila immunity has been the focus of intense study and has impacted other research fields including innate immunity and agriculturally or epidemiologically relevant investigations of insect pests and vectors. Unsurprisingly for such a large body of work, some published results were later found to be irreproducible. Although some results have been contradicted in the literature, many have no published follow-up, either due to a lack of research or low motivation to publish negative or contradictory results. We have addressed this by performing a reproducibility project that analyses the verifiability of claims from articles published on Drosophila immunity before 2011. To assess reproducibility, we extracted claims from 400 articles on the Drosophila immune response to bacteria and fungi and performed preliminary verification by comparing these claims to other published literature in the field. Using alternative approaches, we also experimentally tested some ‘unchallenged’ claims, which had no published follow-up. The intent of this analysis was to centralize evidence on insights and findings to improve clarity for scientists that may base research programs on these data. All our data are published on a publicly available website associated with this article ( https://ReproSci.epfl.ch/ ) that encourages community participation. This article provides a short summary of claims that were found to have contradictory evidence, which may help the community to assess past findings on Drosophila immunity and improve clarity going forward.

Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate mRNA would be an advantageous approach to correct gene expression, but has not been evaluated in an in vivo disease model. Here, we investigated if a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identifiy and engineere human proteins capable of increasing mRNA translation using the CRISPR-Cas Inspired RNA-targeting System (CIRTS) platform to enable programmable, guide RNA (gRNA)-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3), that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold - key phenotypic indicators of Dravet syndrome. This work validates a new strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential translational activation has to address neurological haploinsufficiency.

Membrane contact sites (MCS) between organelles maintain the proximity required for controlled exchange of small molecules and ions yet preventing fusion events that would compromise organelles’ identity and integrity. Here, by investigating the intracellular fate of the disease-causing Z-variant of alpha1 antitrypsin (ATZ), we report on a novel function of MCS between the endoplasmic reticulum (ER) and RAB7/LAMP1-positive endolysosomes in ER-to-lysosome-associated degradation (ERLAD). For this function, the VAPA:ORP1L:RAB7 multi-protein complex forming MCS between the ER and endolysosomes engages, in an ERLAD client-driven manner, the misfolded protein segregation complex formed by the lectin chaperone Calnexin (CNX), the ER-phagy receptor FAM134B and the ubiquitin-like protein LC3. Generation of this supramolecular complex facilitates the membrane fusion events regulated by the SNARE proteins STX17 and VAMP8 that ensure efficient delivery of ATZ polymers from their site of generation, the ER, to the site of their intracellular clearance, the degradative RAB7/LAMP1-positive endolysosomes.

Abstract Understanding how neurons integrate into developing circuits and contribute to functional activity is essential for decoding brain development and plasticity. However, current methods to study neuronal integration often suffer from low throughput, limited spatiotemporal resolution, or invasive procedures that hinder in vivo functional analysis. To overcome these challenges, we present a birthdate-labeling strategy, named CHLOK, based on HaloTag technology and a broad palette of fluorescent synthetic dyes. This approach enables precise multicolor labeling of neurons according to their maturation stage and allows flexible integration into functional assays through compatibility with calcium imaging and optogenetics. We validated CHLOK by mapping birthdate-resolved neuronal activity in the developing visual and motor systems of zebrafish larvae. Our results reveal distinct functional contributions of early- versus late-born neurons, providing new insights into the temporal dynamics of circuit formation. Furthermore, we demonstrate the versatility of this approach, showcasing age-specific multicolor calcium and voltage imaging as well as optogenetic manipulation. By overcoming key limitations of existing techniques, CHLOK offers a powerful, versatile and non-invasive tool for studying neural integration, circuit development and function in vivo. These authors contributed equally: Matthieu Tuffery, Amna Saleem,& Lixia Zhang. These authors jointly supervised this work: Filippo Del Bene & Minoru Koyama.

ABSTRACT Mutations in the innate immune receptor NOD2 are the greatest single genetic risk factors for Crohn’s disease, yet the mechanisms by which NOD2 regulates intestinal homeostasis remain unclear. Here, we used a CRISPR-generated zebrafish model to determine the impacts of NOD2 deficiency on intestinal health. In a series of cellular, molecular, and transcriptomic studies, we uncovered substantial effects of NOD2 deficiency on epithelial and immune compartments, including deregulated expression of developmental pathways critical for establishment and maintenance of the gut epithelium, and an unexpected increase in the expression of multiple estrogen-response genes. In a series of functional assays, we uncovered a mechanistic link between estrogenic signals and NOD2-deficiency phenotypes, whereby exposure to estrogen alone replicated NOD2-deficiency phenotypes, and exposure to an estrogen receptor antagonist reverted the epithelial defects observed in NOD2 mutants. Our findings identify a critical NOD2-estrogen regulatory axis in the establishment of intestinal homeostasis and suggest that hormonal signaling may contribute to the sex-specific pathogenesis of Crohn’s disease.

Pathogens counteract central nodes of NLR immune receptor networks to suppress immunity. However, the mechanisms by which pathogens hijack helper NLR pathways are poorly understood. Here, we show that an effector from the potato late blight pathogen Phytophthora infestans bridges the host protein NbTOL9a, a putative member of the host ESCRT pathway, to a helper NLR to suppress immunity. In this work, we solved the crystal structure of the RXLR-LWY effector AVRcap1b in complex with the ENTH domain of NbTOL9a. The structure revealed that unlike other RXLR-LWY effectors, AVRcap1b has a novel L-shaped fold that defines a new structural family of effectors in the Phytophthora genus. Moreover, we defined the AVRcap1b/NbTOL9a binding interface and designed effector mutants that don’t bind NbTOL9a, impairing immune suppression. This indicates that ENTH binding is required for full virulence activity of this effector. Lastly, we show that AVRcap1b associates specifically with activated NbNRC2 independently of NbTOL9a binding. This suggests that the effector functions as a bridge that interconnects NbNRC2 with the NbTOL9a pathway. These results illustrate an unprecedented pathogen mechanism to hijack helper NLR pathways and suppress immunity.

Background The emergence and spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis ( Mtb ) urge the development of novel drugs and efficient therapeutic programs. A recent study aiming to uncover differential beta-lactam susceptibility phenotypes in clinical strains of Mtb found that the M237V substitution in cwlM ( Rv3915 ) was associated with increased susceptibility to amoxicillin. Considering that Mycobacterium smegmatis ( Msm ) is a widely used surrogate model for Mtb , we constructed a cwlM knockdown mutant in Msm using CRISPR interference (CRISPRi) to elucidate the role of CwlM in beta-lactam susceptibility and intracellular survival. Results Quantitative RT-PCR assays confirmed the successful repression of cwlM , while the phenotyping assays confirmed the essentiality of CwlM-related processes for mycobacterial viability. Collectively, the antibiotic susceptibility assays suggested that CwlM SMEG may promote beta-lactam resistance, particularly to meropenem and cefotaxime. Moreover, CwlM SMEG was found to support M. smegmatis intracellular survival within THP-1-derived macrophages. To address conflicting reports regarding its predicted peptidoglycan (PG) hydrolase activity, we purified recombinant CwlM TB . The Micrococcus luteus -derived PG-based zymogram indicated that CwlM TB lacks PG-hydrolytic activity, suggesting it might act as a regulator of PG biosynthesis instead. Conclusions Our findings indicate that CwlM contributes to beta-lactam resistance and intracellular survival, regardless of lacking detectable PG-hydrolytic activity. Overall, CwlM was found to be essential and highly vulnerable, highlighting its potential as a therapeutic target that warrants further investigation.

Jumbo phages protect their genomes from DNA-sensing bacterial defense systems by enclosing them within vesicles and nucleus-like compartments. Very little is known about defense systems specialized to counter these phages. Here, we show that AVAST Type 5 (Avs5) systems, part of the STAND superfamily and spanning three phylogenetic Avs5 clades, confer conserved immunity against jumbo phages. Using localization microscopy and biotin proximity labeling we demonstrate that Avs5 localizes to early infection vesicles, where it senses an essential, early expressed phage protein named JADA—Jumbophage AVAST5 Defense Activator. Recognition of phage infection triggers the Sir2-like effector domain of Avs5 across all three Avs5 clades, resulting in rapid NAD + hydrolysis, disruption of phage nucleus formation, and arrest of infection. These findings reveal a spatially coordinated bacterial immune strategy that targets an early vulnerability in jumbo phage infection.

Abstract Despite its remarkable clinical success in human cancer treatment, immune checkpoint blockade is effective only in a minority of patients. One major obstacle is tumor-driven impairment of T cell priming and early activation, however, the underlying mechanism remains elusive. Here we identify TMEM176B-positive exosomes specifically secreted by cancer cells in plasma of cancer patients rather than healthy donors, high levels of which correlate with worse prognosis and unfavorable outcomes of anti-PD1 therapy. A small-scale CRISPR-Cas9 screen discovers Tmem176b on tumor-derived exosomes as a negative regulator of T cell early activation. Genetic ablation of Tmem176b in mouse cancer cells substantially suppresses tumour growth in a CD8 + T cell-dependent manner. Mechanistically, tumour-derived exosomal Tmem176b attenuates proximal T cell receptor signaling in CD8 + T cells by recruiting tyrosine phosphatase Shp1 to immunological synapse. Blocking TMEM176B, either using neutralizing antibody or using competitive peptide to disrupt Tmem176b-Shp1 interaction, remarkably restrains tumour progression in models of mouse and human cancers, and synergizes with anti-PD1/PD-L1 therapy. Our findings not only uncover tumor-derived Tmem176b as a promising target for cancer immunotherapy, but also provide a potential non-invasive diagnostic tool to detect early cancer and predict clinical response to anti-PD1 therapy.

ABSTRACT DNA polymerase theta (Polθ) plays a crucial role in the repair of DNA double-strand breaks (DSBs) by microhomology-mediated end joining (MMEJ). We previously demonstrated that Polθ inhibition (Polθi) is an effective and well-tolerated approach to sensitise tumours to radiotherapy (RT). Here, we profiled 54 cancer cell lines and found that Polθi induces significant radiosensitisation in most models, though with marked variability not explained by indicators of Polθ activity. To pinpoint molecular determinants of radiosensitisation by Polθi, we performed a CRISPR knockout screen which revealed loss of the TP53BP1/Shieldin pathway component SHLD2 ( FAM35A ) as a vulnerability to Polθi combined with RT. We demonstrated that SHLD2 loss not only increases sensitivity to RT alone, but also enhances the radiosensitising effect of Polθi, both in vitro and in vivo . Importantly, we found that SHLD2 is deleted in a subset of human prostate cancers, often co-occurring with PTEN loss, an adverse prognostic factor. Furthermore, we show that SHLD2 -deficient cancer cells are more reliant on Polθ to prevent DSB accumulation and chromosomal instability. In summary, we discovered SHLD2 loss as a novel collateral vulnerability that can be exploited through combined treatment with Polθi and RT.