Background: The emergence and spread of antimicrobial resistance (AMR) genes in environmental microbiomes pose a critical threat to global health security. Current detection methods are time-consuming and often lack the sensitivity required for early environmental surveillance. Methods We developed a novel CRISPR-Cas13a-based biosensor system coupled with isothermal amplification for rapid, field-deployable detection of clinically relevant AMR genes (blaNDM-1, mcr-1, and vanA) in environmental samples. The system integrates microfluidic sample processing with fluorescent readout and smartphone-based detection. Results Our biosensor demonstrated exceptional sensitivity with detection limits of 10 copies/μL for target AMR genes, achieving 100% specificity against non-target sequences. Field testing across 150 environmental samples from wastewater treatment plants, agricultural runoff, and hospital effluents revealed previously undetected AMR hotspots. The system provided results within 45 minutes compared to 72 hours for conventional PCR-based methods. Longitudinal monitoring revealed seasonal fluctuations in AMR gene prevalence, with peak concentrations coinciding with agricultural antibiotic usage patterns. Conclusions This innovative biosensor platform enables rapid, sensitive detection of AMR genes in environmental settings, providing a powerful tool for real-time surveillance and early warning systems. The technology addresses critical gaps in current AMR monitoring capabilities and offers significant potential for global implementation in resource-limited settings.

Abstract Autosomal dominant tubulointerstitial kidney disease -UMOD (ADTKD-UMOD) is characterized by progressive renal interstitial inflammation and fibrosis. However, its underlying mechanisms remain unclear. Here, we identify a large ADTKD pedigree harboring a novel UMOD p.H36Y mutation. Using CRISPR/Cas9 technology, we generated a UmodH36Y/+ mouse model that recapitulates the key phenotypes observed in affected individuals, including renal dysfunction, cyst formation, interstitial inflammation, and fibrosis. Multi-omics analyses revealed marked macrophage pyroptosis in UmodH36Y/+ kidneys. Treatment with disulfiram (DSF), a pyroptosis inhibitor, significantly alleviated interstitial inflammation and improved renal function. Mechanistically, the Umod p.H36Y variant activated the amyloid precursor protein (App)-Cd74 axis which mediated the crosstalk between renal TECs and macrophages. This axis sustains NF-κB pathway activation in macrophages, initiating pyroptosis and pro-inflammatory cytokine release. Disrupting App-Cd74 signaling effectively suppressed macrophage pyroptosis. Notably, pharmacologic inhibition using ARN2966, a small-molecule App inhibitor, markedly attenuated renal injury in UmodH36Y/+ mice. Collectively, these findings uncover a novel, targetable pathway in ADTKD-UMOD.

GPR132 (G2A), a lipid- and pH-sensing GPCR, has been implicated in both pro- and anti-inflammatory signaling, but its in vivo function in wound repair and infection control remains unknown. Here, we investigated the role of GPR132b, a zebrafish homolog of G2A, in regulating innate immune responses. Using CRISPR-Cas9, we generated gpr132b mutants and found that they exhibit enhanced wound healing following sterile injury but increased susceptibility to Listeria monocytogenes infection, indicating that GPR132b modulates a trade-off between wound repair and antimicrobial defense. The enhanced regrowth phenotype was associated with increased macrophage accumulation at the wound site and reduced basal expression of the pro-inflammatory cytokine tnf-α . Macrophage depletion suppressed the enhanced regrowth phenotype, suggesting a functional role for macrophages in GPR132b-mediated repair. Pharmacological inhibition of cyclooxygenase (COX) and 12-lipoxygenase (12-LOX) pathways mimicked the gpr132b mutant phenotype in wild-type larvae, indicating that GPR132b likely responds to lipid-derived signals. Together, our findings reveal that GPR132b acts as a c ontext-dependent regulator of innate immunity, impairing efficient tissue repair in sterile conditions while supporting pathogen resistance during infection. Our results underscore the importance of GPCR-mediated signaling in orchestrating effective responses to tissue injury and infection.

Twenty causative genes have been reported that cause non-syndromic childhood glaucoma associated with anterior segment dysgenesis. FOXC1, PAX6 and PITX2 are the most well-known, but cases linked to SLC4A11, PITX3 and SOX11 have also been reported. As genetic testing becomes increasingly widespread and rates of molecular diagnosis rise, the extent of phenotypic overlap between the different genetic causes of non-syndromic glaucoma associated with anterior segment dysgenesis is becoming more evident. Taking aniridia as an example, whilst PAX6 mutations remain the predominant cause, variants in CYP1B1, FOXC1, PXDN and SOX11 have also been reported in patients with childhood glaucoma and aniridia. Developments in molecular-based therapies for retinal and corneal disease are advancing rapidly, and pre-clinical studies of gene-based treatments for glaucoma and aniridia are showing promising results. Use of adeno-associated viral vectors for gene delivery is most common, with improvements in intraocular pressure and retinal ganglion cell survival in Tg-MYOCY437H mouse models of glaucoma, and successful correction of a germline PAX6G194X nonsense variant in mice using CRISPR/Cas9 gene editing. This review will explore the actions and interactions of the genetic causes of non-syndromic glaucoma associated with anterior segment dysgenesis and discuss current developments in molecular therapies for these patients.

The nuclear pore complex (NPC) forms a large channel that spans the double lipid bilayer of the nuclear envelope and is the central gateway for macromolecular transport between the nucleus and cytoplasm in eukaryotes. NPC biogenesis requires the coordinated assembly of over 500 proteins culminating in the fusion of the inner and outer nuclear membranes. The molecular mechanism of this membrane fusion step that occurs in all eukaryotes is unknown. Here, we elucidate the mechanism by which two paralogous transmembrane proteins, Brl1 and Brr6, mediate membrane fusion in S. cerevisiae. Both proteins form multimeric, ring-shaped complexes with membrane remodeling activity. Brl1 is enriched at NPC assembly sites via a nuclear export sequence and then interacts with Brr6 across the nuclear envelope lumen through conserved hydrophobic loops. Disrupting this interaction blocks fusion and halts NPC assembly. Molecular dynamics simulations suggest that the Brl1-Brr6 complex drives membrane fusion by forming a channel across bilayers that enables lipid exchange. Phylogenetic analyses reveal that Brl1/Brr6 homologues are broadly distributed across eukaryotes, and functional experiments in human cells and D. melanogaster establish CLCC1 as an NPC fusogen in metazoans. Together, our results uncover a novel, conserved mechanism for membrane fusion in eukaryotes.

Trypanosoma cruzi , the causative agent of Chagas disease, relies on complex gene regulatory mechanisms to adapt to its diverse host environments. Although this parasite lacks canonical transcriptional control, epigenetic regulation plays a pivotal role in modulating gene expression. Bromodomain-containing factors (BDFs), known for recognizing acetylated lysines on histones, have emerged as key regulators of chromatin structure and gene activity. Among the eight predicted BDFs in T. cruzi, Tc BDF6 is part of a TINTIN-like complex with Tc MRGx and Tc MRGBP, homologous to components of the NuA4/TIP60 chromatin remodeling complex. To investigate the function of Tc BDF6, we generated knockout (KO) parasites using CRISPR/Cas9 gene editing. While BDF6-deficient epimastigotes did not exhibit very significative growth differences, the resulting metacyclic trypomastigotes displayed drastically reduced infectivity. Strikingly, once inside host cells, Tc BDF6 deficient parasites differentiated into amastigotes but failed to replicate. This intracellular arrest was partially reversed by episomal re-expression of Tc BDF6. Consistently, BDF6 KO parasites also exhibited impaired infectivity, a defect that was also rescued in the add-back line. Our findings highlight Tc BDF6 as a critical regulator of intracellular parasite development, operating in stages beyond epimastigotes where epigenetic plasticity is essential for host adaptation. The unique, stage-specific phenotype of BDF6 knockouts underscores its functional importance and suggests that bromodomains may represent novel therapeutic targets against T. cruzi . Author Summary To survive in the dramatically different environments of its life cycle, Trypanosoma cruzi , the parasite that causes Chagas disease, must finely tune its gene expression. Unlike many organisms, T. cruzi lacks classical transcriptional regulation and instead relies on epigenetic mechanisms. Among the proteins involved in this regulation are bromodomain containing factors, which interpret chemical marks on chromatin. In this study, we focused on Tc BDF6, a bromodomain protein thought to be part of a chromatin remodeling complex. Using CRISPR/Cas9, we disrupted the TcBDF6 gene and discovered that, while the mutant parasites could still form infective stages, they failed to replicate inside host cells. Reintroducing TcBDF6 , it was rescued this defect, confirming its critical role in intracellular development. Our findings highlight the importance of epigenetic control in parasite survival and suggest that bromodomain proteins could be valuable targets for future treatments against Chagas disease.

ABSTRACT The TTC22 gene encodes a protein containing seven tetratricopeptide repeats (TPRs), which mediate protein‒protein interactions as chaperones. We previously reported that the level of TTC22 transcript variant 1 ( TTC22v1 ) was downregulated in human colon adenocarcinoma (COAD) and that TTC22 upregulated m6A-mediated WTAP and SNAI1 expression via the TTC22‒RPL4 interaction and subsequently promoted COAD metastasis. Thus, a commercially available C57BL/6N mouse model in which the Ttc22 exon 2&3 encoding the TPR4 (equal to the human TTC22 TPR3) domain was knocked out via CRISPR-Cas9 was used to evaluate the contribution of Ttc22 to the development of mice and COAD. Unfortunately, the long-term observation results demonstrated that Ttc22 knockout (KO, including Ttc22 -/+ or Ttc22 -/- ) did not affect the body weight, development, fertility, or spontaneous tumor incidence of male or female mice. No differences in the incidence of AOM/DSS-induced COAD were observed between these mouse groups, although Ttc22 exon 2&3 deletion partially resulted in the upregulation of adaptive response genes in the colon mucosa. Further study revealed that TPR4 deletion did not disrupt the effect of TTC22 on WTAP upregulation. Consistently, TPR4 deletion did not affect the abundance of total RNA m6A in the colon tissues of the mice, suggesting that the TPR4-deleted Ttc22 mutant remains functional. In conclusion, the TPR4 domain is not essential for the Ttc22 protein. Loss of Ttc22 TPR4 caused no observable changes in the development of C57BL mice or their susceptibility to treatment with the chemical carcinogen AOM/DSS. Whether an essential sequence for a target gene is knocked out should be carefully evaluated before a gene knockout model is employed in formal experiments. Highlights Knockout of Ttc22 exons 2 and 3 does not affect the development of C57BL mice. Ttc22 knockout does not affect the induction of mouse colon cancer by AOM/DSS. Loss of the Ttc22 exons 2 and 3 cannot disrupt the functions of TTC22.

Summary Brown algae represent the third most complex lineage to evolve multicellularity, independently from plants and animals. However, functional studies of their development, evolution, and biology have been constrained by the lack of efficient and scalable genome editing tools. Here, we report a robust, high-efficiency, and transgene-free CRISPR–Cas12-based genome editing method applicable across four ecologically and biotechnologically important brown algal species. Using Ectocarpus as a model, we optimized a PEG-mediated RNP delivery system employing a temperature-tolerant Cas12 variant, achieving reproducible, high-efficiency editing across multiple loci without the need for cloning or specialized equipment. As proof of concept, we precisely recapitulated the hallmark imm mutant phenotype by editing the IMMEDIATE UPRIGHT (IMM) locus, a phenotype previously described only from a rare spontaneous mutation. APT/2-FA-based selection further improved specificity with minimal false positives. The protocol was readily transferrable to other species, including kelps long considered recalcitrant to transformation. This platform now makes functional genomics accessible in brown algae, enabling mechanistic dissection of developmental processes, life cycle transitions, and the independent origins of complex multicellularity. Our work enables the broader adoption of brown algae as experimental models and provides a valuable platform for marine biotechnology and evolutionary research. Motivation Although most of biodiversity on Earth lives in oceans, a significant proportion of its organisms remain largely uncharacterized. Brown algae represent one of such understudied group of marine photosynthetic eukaryotes. Despite their importance as emerging models for developmental evolution and blue biotechnology, functional genomics in brown algae has remained largely inaccessible due to a lack of efficient and scalable genome editing tools. Our aim is to democratize genome editing in brown algae by developing a high-efficiency, transgene-free protocol that works across multiple species, without the need for specialized equipment. This high-efficiency method fully enables the field of functional genomics in an unexplored multicellular lineage. Highlights High-efficiency, low-cost genome editing in brown algae without specialized equipment. ·Applicable to non-model species, including those of economic importance.

Abstract Culex mosquitoes transmit major pathogens including West Nile virus, encephalitis, filariasis, and avian malaria, threatening public health, poultry, and ecosystems. We engineered a CRISPR-based population suppression gene drive targeting a conserved exon of the doublesex ( dsx ) gene. The drive incorporates a recoded dsxM segment to preserve male function while converting genetic females into sterile intersexes, enabling male-biased propagation and removal of fertile females. It achieves super-Mendelian inheritance (~ 71%) and generates partially dominant sterile resistance alleles via end-joining, resulting in intersex phenotypes with reduced fertility and hatchability. Modeling predicts that this RIDD (Release of Insects carrying a Dominant-sterile Drive) system can suppress populations at low intrinsic growth rates and release ratios, outperforming SIT and fs-RIDL strategies in persistence and efficiency, with further gains achievable through improved cleavage rates. This study establishes a self-limiting gene drive framework for Culex suppression, highlightling the potential of targeting conserved sex-determination pathways for sustainable vector control.

Profiling antigens presented on MHC class I molecules on the cell surface is essential to identify candidate antigens for targeted and personalized immunotherapies. However, mass spectrometry-based immunopeptidomics has traditionally been limited by high input requirements, extensive sample manipulation, and expensive reagents. To overcome these challenges, we developed MHC1-TIP: a scalable, single-tube and cost-effective workflow to enable robust MHC-I ligandome recovery from cell lines, patient-derived organoids, and sub-milligram amounts of clinical tissues. Moreover, MHC1-TIP also preserves compatibility with additional omics profiling technologies and we demonstrate its capacity for quantitative and multimodal profiling of the proteome and immunopeptidome from the same sample to enable integrated analyses of protein expression and antigen presentation. Application of MHC1-TIP to primary renal cell carcinoma fragments revealed extensive intratumoral heterogeneity in antigen presentation that was poorly correlated with source protein expression. MHC1-TIP represents a broadly applicable and sensitive approach for low-input, multimodal immunopeptidomics with clinical and translational relevance.

Maintaining intestinal homeostasis relies on the intricate interplay among the mucosal epithelium, immune system, and host microbiome. A key question is how innate immune cells sense and process microbes in the gut lumen, eliciting appropriate protective responses without causing tissue injury. Clearance of invading microbes and initiation of downstream inflammatory responses are central to this process and require proper function of the endolysosomal system. Dysfunction of this system can predispose the host to chronic inflammatory disorders and acute infections. Here, through forward genetic screening of N-ethyl-N-nitrosourea (ENU)-mutagenized mice and CRISPR/Cas9 validation, we identify Lrmda , encoding leucine-rich melanocyte differentiation-associated protein (LRMDA), as a key regulator of intestinal homeostasis. Using hematopoietic chimera and conditional knockouts, we show that LRMDA functions primarily in CD11c + cells, including mucosal dendritic cells (DCs) and macrophages, but not in non-hematopoietic cells. Proteomic, cellular, and biochemical analyses reveal that LRMDA directly and cooperatively interacts with the endolysosome-specific small GTPase Rab32 and the endosomal recycling complex Retriever. Loss of LRMDA or Retriever function increases susceptibility to dextran sodium sulfate (DSS)-induced colitis and impairs clearance of Listeria monocytogenes . Together, our findings establish the Rab32-LRMDA-Retriever complex as a critical regulator of endolysosomal trafficking in innate immune cells, essential for maintaining intestinal immune homeostasis.

ABSTRACT BACKGROUND While mitochondrial dysfunction clearly drives cardiac deterioration in major heart diseases, the mechanisms controlling mitochondrial quality remain incompletely understood. This study investigated whether TIGAR (TP53-induced glycolysis and apoptosis regulator) deficiency influences cardiac protection through mitochondrial quality control pathways. METHODS We generated both whole-body and cardiomyocyte-specific TIGAR knockout mice that were assessed for cardiac function following myocardial infarction (induced by left anterior descending coronary artery ligation) and diet-induced cardiomyopathy (using a 6-month high-fat diet protocol). Mitochondrial quality control was evaluated through mitophagy assays, subcellular fractionation, and molecular analyses. Epigenetic regulation was assessed using whole-genome bisulfite sequencing, chromatin immunoprecipitation, and CRISPR-mediated gene editing in multiple cell lines. RESULTS Both whole-body (TKO) and cardiomyocyte-specific (hTKO) TIGAR knockout mice demonstrated cardioprotection following myocardial infarction. These animals maintained significantly better ejection fraction (43.35±17.76% vs 26.36±9.83% in wild-type controls, P<0.05) and displayed complete resistance to diet-induced cardiac hypertrophy, despite comparable weight gain. TIGAR deficiency led to dramatic increases in Parkin expression across multiple tissues, 6-fold increases in heart and muscle, and 5-fold increases in brain, which enhanced mitophagic responses during metabolic stress conditions including fasting and high-fat diet feeding. Generation of Parkin/TIGAR double knockout mice eliminated this protection, confirming Parkin’s essential role. Notably, adult manipulation of TIGAR through viral overexpression or knockdown failed to alter Parkin levels, suggesting developmental programming. Whole-genome bisulfite sequencing revealed reduced DNA methylation in a specific 3.2 kb region within Parkin gene intron 10, and CRISPR deletion of this regulatory region increased Parkin expression 10-fold in C2C12 myoblasts and 6-fold in 3T3-L1 fibroblasts. CONCLUSIONS These findings reveal a novel TIGAR-Parkin regulatory axis operating through epigenetic mechanisms during cardiac development to establish lifelong cardioprotection via enhanced mitochondrial quality control. This discovery points toward new therapeutic approaches targeting developmental metabolic programming for heart disease prevention and identifies specific epigenetic targets for cardiovascular therapy. CLINICAL PERSPECTIVE What Is New? TIGAR deficiency establishes lifelong cardiac protection through developmental epigenetic programming of Parkin expression. A novel 3.2 kb differentially methylated region in Parkin intron 10 regulates mitochondrial quality control in the heart. Early metabolic programming during cardiac development can establish permanent cardioprotective phenotypes. The TIGAR-Parkin axis provides protection against both acute ischemic injury and chronic metabolic cardiomyopathy. What Are the Clinical Implications? Targeting the TIGAR-Parkin pathway could provide novel therapeutic approaches for preventing both ischemic heart disease and diabetic cardiomyopathy. Early developmental interventions targeting cardiac metabolism might establish lifelong cardiovascular protection. Epigenetic modifications of mitochondrial quality control genes represent potential therapeutic targets. The findings suggest optimal timing for cardiovascular preventive interventions may be during critical developmental windows.

The entomopathogenic nematodes (EPN) of the genus Steinernema serves as a valuable experimental model for studying microbial symbiosis and is economically important as organic pest control agent in agriculture. Although most Steinernema species are dioecious (male-female), consistent genetic manipulation has thus far only been demonstrated in the hermaphroditic species Steinernema hermaphroditum . In this study, we adapted a CRISPR-Cas9–based gene editing approach to Steinernema feltiae , a dioecious species widely used in agricultural applications. Using gonadal microinjection, we targeted the conserved gene unc-22 in S. feltiae and generated stable mutant lines with large on-target deletions. Mating tests revealed that Sf-unc-22 is X-linked and exhibits a conditionally dominant twitching phenotype. Additionally, Sf-unc-22 mutants display distinct body morphology compared to wild-type nematodes. Homozygous mutant lines can be reliably maintained through cryopreservation. Altogether, our work provides a proof of concept that genetic tools developed in S. hermaphroditum can be effectively adapted to other agriculturally relevant and dioecious Steinernema species— broadening the scope of molecular genetic research in microbial ecology and enhancing their potential applications in agriculture.

During development, neurons are produced in excess and those that receive trophic support are maintained, whereas excess neurons are eliminated, enabling the establishment of appropriate neural circuits. In vertebrates, neurotrophin ligands promote cell survival during periods of naturally occurring cell death, by signalling through p75 and Trk receptors. In the Drosophila optic lobe, a wave of apoptosis occurs during neural circuit development; however, whether this also involves neurotrophism remains unresolved. Drosophila neurotrophins (DNTs) are encoded by spätzle (spz) paralogue genes and bind Toll receptors instead. Here, we focused on DNT-3 (previously known as spz-3) and DNT-2 (also known as spz-5 ) to ask whether they underlie neurotrophism in visual system development. We show that DNT-3 ( spz-3 ) and DNT-2 ( spz-5 ) are both expressed in the retina and in medulla neurons, and multiple Tolls are expressed across lamina and medulla neurons. Over-expression of DNT-3 ( spz-3 ) and DNT-2 ( spz-5 ) could rescue natural occurring cell death, whereas their loss of function caused cell death, showing that DNT-3 and DNT-2 can, and are required to, promote cell survival during optic lobe development. Importantly, DNT-2 is expressed in Mi1 neurons and Toll-2 in connecting L1 neurons. We show that DNT-2 functions in concert with Toll-2, as Toll-2 RNAi knock-down prevented the rescue of apoptosis by DNT-2 over-expression and all Toll-2+ neurons were lost in DNT-2 mutants. Furthermore, alterations in DNT-2 or Toll-2 expression levels impaired connectivity of L1 neurons at the M1 medulla layer and altered dendritic morphology of L1 neurons. These data suggest that L1 neurons could take up DNT-2 secreted from medulla neurons during the establishment of connectivity patterns. As DNT-3 ( spz-3 ) and DNT-2 ( spz-5 ) are expressed in the medulla and they could influence both lamina and medulla neurons, this suggests that their function maintaining cell survival could enable the stabilisation or alignment of connected neurons across medulla columns.

ABSTRACT Doyne honeycomb retinal dystrophy is an incurable juvenile macular dystrophy that leads to visual impairment by early to mid-adulthood. It is an autosomal dominant disorder caused by a c.1033T>C, p.Arg345Trp variant in EFEMP1 , and is characterised by the early onset extracellular deposition of drusen between the retinal pigment epithelium basement membrane and underlying layers of Bruch’s membrane. In this study, we developed an antisense oligonucleotide approach to target EFEMP1 . We reprogrammed patient-derived renal epithelial cells to induced pluripotent stem cells followed by directed differentiation to retinal pigment epithelium and compared the phenotype to gene-corrected and EFEMP1 knockout patient-derived retinal pigment epithelium. In the patient-derived disease model, remodelling of the extracellular matrix occurred with progressive accumulation of extracellular deposits containing the drusen-associated proteins apolipoprotein E and collagen IV, in addition to EFEMP1. Moreover, the intracellular accumulation of neutral lipids was evident. We developed an allele-specific antisense oligonucleotide which specifically and effectively promoted the clearance of the EFEMP1 c.1033T>C transcript in the patient-derived disease model following assisted or gymnotic delivery. In this disease model, gymnotic delivery led to a decrease in extracellular deposits and cleared the intracellular accumulation of lipids, even after the onset of this disease phenotype, suggesting this could be a practical and effective therapeutic approach.

Development of scalable and highly adaptable platforms to characterise high consequence infections are essential for limiting the impact of future viruses with pandemic potential. As part of this, an understanding of the virus-host interactions is vital, with the cell entry mechanism being crucial for the development of therapeutics and vaccines. Current approaches in this field depend on assays with authentic (live) viruses that require high containment facilities. However, these are hindered by high costs, need for highly trained staff, slow processing times and limited scalability. Using pseudotyped viruses (PV) expressing the chikungunya (CHIKV) envelope protein as a proof-of-principle, we developed a novel screening platform, Ceudovitox , to identify cellular factors involved in viral entry. PVs were engineered to express the herpes simplex virus-1 thymidine kinase, which following addition of ganciclovir, can selectively kill infected cells. Then a heterogenous pool of knockout cells were produced using the CRISPR-Cas9 library. Infection of these cells with the “killer” PV system permitted positive selection of cells refractory to viral infection and, through next generation sequencing, identification of a number of factors involved in CHIKV entry. Matrix metalloproteinases were identified as novel entry factors and demonstrated that MMP-targeting drugs efficiently inhibit PV and authentic CHIKV infections. These results suggest this platform holds great promise as a pandemic-preparedness tool that increases the capacity and speed of screening for cell entry factors. It is also a safe platform able to dissect the gene network involved in virus entry. Ceudovitox will help identify new therapeutic targets, thereby aiding the development of treatments against future outbreaks or pandemic pathogens and making a significant contribution to the “100 days” mission. Author Summary The increased threat of the emergence of new viral pathogens with pandemic potential highlights the importance of developing scalable high-throughput screening platforms to quickly characterise new emergent viral pathogens. Improving understanding of virus-host interactions, including how viruses enter cells, can help fast development of new targeted therapeutics and reduce pandemic burdens. Current approaches rely on using ‘live’ viruses, which are highly infectious and must be manipulated in high containment facilities, which are slow and cumbersome. Our new screening platform, Ceudovitox , is a safer, faster and more scalable alternative. Through using pseudotyped viruses, which can only undergo one replication cycle, we are able to distinguish virus entry factors of high containment viruses within highly abundant low containment facilities, allowing us to quickly characterise potential entry factors of high containment viruses. Within this study, Ceudovitox was shown to recognise both known and novel entry factors of Chikungunya virus; a virus which causes chronic arthralgia and arthritis. Matrix metalloproteinases were amongst novel entry factors identified and were seen to reduce virus infection in both pseudotyped and authentic virus assays, demonstrating how Ceudovitox can correctly discover virus entry factors of ‘live’ high containment viruses. Ceudovitox’s utility as a screening platform for high containment viruses and new emerging pathogens, has the potential increase understanding and speed up the development of new therapeutics during pandemics, helping to reach the ‘100 Day Mission’ of pandemic preparedness.

Somatic mutations in ribosomal proteins (RPs), including RPL5, have been reported in approximately 10% of pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL). In cancer, the incorporation of mutant RPs into ribosomes often disrupts canonical ribosome function, thereby contributing to disease development. In this study, we aimed to characterize the effects of the RPL5-I60V mutation in the context of T-ALL, focusing on its impact on translation and cellular responses to a panel of compounds in vitro. Using CRISPR-Cas9, we generated a homozygous knock-in mutant in Jurkat cells and investigated its effects on ribosome biogenesis. We observed both quantitative and qualitative alterations in the production of the large ribosomal subunit. Ribosomes containing the mutant RPL5 protein exhibited intrinsically increased protein synthesis activity, which correlated with enhanced cellular proliferation. We then evaluated the response of these mutant cells to a panel of compounds targeting protein synthesis at various levels—including an MNK1 inhibitor, metformin, silvestrol, homoharringtonine, anisomycin, resveratrol, and hygromycin B—as well as cytarabine, a chemotherapeutic agent commonly used in T-ALL treatment. Our results showed that the RPL5-I60V mutation confers increased sensitivity to most of these compounds, with the exception of hygromycin B. This study advances our understanding of how oncoribosomes contribute to cancer pathogenesis and highlights the therapeutic potential of directly or indirectly targeting altered ribosomes, offering insights for the development of personalized treatment strategies. Highlights RPL5 mutations have been reported in T-ALL Ribosomes containing the RPL5 I60V mutation exhibit increased translational activity Increased proliferation is observed in cells harboring the RPL5-I60V mutation RPL5-I60V confers a specific drug sensitivity profile Statements and Declarations Competing interests: The authors declare that they have no conflict of interest.

Abstract Ribosomal DNA (rDNA) double-strand breaks (DSBs) threaten genome integrity due to the repetitive and transcriptionally active nature of rDNA. The nucleolus, while central to ribosome biogenesis, also functions as stress sensor. Here, we identify a transcription-dependent mechanism in which RNA polymerase II (RNAPII) is essential for homologous recombination (HR) repair of rDNA DSBs. Using CRISPR-induced breaks, high-resolution imaging, and transcriptional inhibition, we show that RNAPII activity drives the formation of nucleolar repair caps. Mechanistically, CtIP promotes RNAPII recruitment and H3K36 trimethylation at rDNA lesions, facilitating HR. Disruption of this RNAPII–CtIP–H3K36me3 axis impairs cap formation and repair, leading to persistent damage. RNAPII inhibition exacerbates genome instability and synergizes with rDNA breaks to induce cancer cell death, without acutely impairing ribosome function. These findings uncover a co-transcriptional mechanism of rDNA repair and highlight RNAPII-mediated chromatin remodeling and spatial reorganization as key to nucleolar genome maintenance and potential targets for cancer therapy.

Summary Emerging research has implicated Alzheimer’s disease (AD) pathology with dysregulation of many key pathways in microglia, including lipid transport and metabolism, phagocytosis of plaques, and lysosomal function. However, the exact mechanisms underlying these pathways remain poorly understood. Leveraging high-throughput CRISPR screens to understand the interplay between these pathways may enable novel therapeutic strategies for AD and other neurological diseases. Here, we constructed activation and interference CRISPRa/i libraries targeting 203 genes, 71 of which were identified through neurodegenerative GWAS, and 132 additional genes linked to microglial functions. We used this library to conduct pooled CRISPRa/i screens across a range of functional assays relating to lipid metabolism and lysosomal function using a monocytic cell line, THP-1. We identified a core set of lipid and lysosome mediators and validated a subset in primary macrophages. To gain insights into transcriptional states modulated by these genes we also applied the CRISPRa/i libraries to Perturb-seq, enabling us to capture transcriptomic changes. Through non-negative matrix factorization, we identified five gene programs altered by our perturbation library. We then used an integrative analysis of functional screen data with Perturb-seq data that enabled us to uncover novel functions and genetic relationships between perturbations. This multidimensional resource links genetic perturbations to phenotypes and transcriptional programs, establishing a scalable framework for systematic gene discovery in neurodegeneration and beyond. Abstract Figure Graphical Abstract

Allelic variation can impact viral clearance and disease severity, but the effect of the autoimmunity-associated allelic variant of Ptpn22 (PEP-R619W) on antiviral immunity remains incomplete. This research defines how the loss of Ptpn22 (PEP-null) and PEP-R619W changes antiviral immunity during acute coronavirus infection. We address the hypothesis that CRISPR/Cas9-generated PEP-null and PEP-R619W mice have enhanced antiviral immunity over PEP-WT mice during coronavirus infection. Following Mouse Hepatitis Virus (MHV) A59 infection, we interrogated pathology, cytokine production, and cellular responses in the blood, spleen, and liver of PEP-WT, PEP-null, and PEP-R619W mice. Key findings show that PEP-R619W mice have reduced viral titer and weight loss, increased survival, and more mature NK cells in the liver and spleen compared to PEP-WT mice. Interestingly, protection against disease in PEP-null mice was inoculation-dose-dependent, whereas PEP-R619W conferred immunity regardless of infection dose. Further, Rag1-/- PEP-R619W mice had increased survival and reduced viral titer over Rag1-/- PEP-WT mice. PEP-R619W mice also had higher concentrations of IFNγ and enhanced IFNγ production by mature NK cells in the liver at 3 days post-infection. Finally, NK cell depletion elevated PEP-R619W viral titer to similar levels as PEP-WT mice. This is one of the first studies investigating the role of Ptpn22 within NK cells and demonstrates that the Ptpn22 allelic variant augments NK cell function and is beneficial during coronavirus infection. One Sentence Summary The Ptpn22 autoimmunity-associated allele uniquely augments innate immunity to protect against acute coronavirus infection.

Tissue-resident macrophages efficiently internalize Aspergillus fumigatus spores, forming a critical first line of defense against infection. However, the mechanisms that these cells use to control spores in vivo remain incompletely defined. Here, we used the live imaging capabilities of the larval zebrafish host model to assess the role of the v-ATPase complex in macrophage-mediated defense against A. fumigatus in a whole vertebrate animal. For the first time we are able to visualize co-localization of A. fumigatus spores with the key v-ATPase subunit Atp6v1h in macrophages inside of an infected animal. As macrophages only have a low ability to kill spores, this co-localization occurs as early as 1-day post-injection and persists for multiple days. Surprisingly, macrophage spore killing is not further reduced by targeting of atp6v1h with CRISPR/Cas9. Instead, v-ATPase deficiency profoundly impacts macrophage-mediated control of spore swelling, decoupling the macrophage functions of spore killing and inhibition of germination. We also identify a role for the v-ATPase complex in macrophage control of extracellular hyphal growth. These effects on macrophage function drive significantly decreased host survival in larvae lacking a functional v-ATPase. We also report broad effects of v-ATPase deficiency on macrophage numbers, apoptosis in the hematopoietic tissue, and potential neutrophil functions, reflecting the importance of this complex in host antifungal immunity. Graphical Abstract Highlights v-ATPase function in macrophages restricts Aspergillus germination and hyphal growth Spore killing by macrophages is independent of v-ATPase function Loss of macrophage v-ATPase mimics the effects of macrophage deficiency in vivo

The transcription factor ATF6 has a central role in adapting mammalian cells to endoplasmic reticulum (ER) stress via the Unfolded Protein Response (UPR). This has driven efforts to identify modulators of ATF6 signalling. Here, an unbiased genome-wide CRISPR-Cas9 screen performed in Chinese Hamster Ovary (CHO) cells revealed that proteolytic processing of the ATF6α precursor to its active form was impaired in CHO cells lacking the ER-resident solute carrier SLC33A1, a transporter involved in acetyl-CoA import, sialylation and Nε-lysine protein acetylation. Cells lacking SLC33A1 constitutively trafficked the ATF6α precursor to the Golgi, but exhibit impaired subsequent Golgi processing, correlating with altered ATF6α Golgi glycosylation. SLC33A1 deficiency also deregulated activation of the IRE1 branch of the UPR, pointing to a selective loss of ATF6α-mediated negative feedback in the UPR. Notably, Slc33a1 -deleted cells accumulated higher levels of unmodified sialylated N-glycans, precursors to acetylated glycans, likely reflecting impaired glycan processing. By contrast, deletion of ER-localised acetyltransferases NAT8 and NAT8B, which catalyse protein Nε-lysine acetylation in the secretory pathway, did not replicate the ATF6α processing defects observed in Slc33a1 -deficient cells. Together, our findings highlight a role for SLC33A1-mediated metabolite transport in the post-ER maturation of ATF6α and point direct links between small-molecule metabolism and branch-specific signalling in the UPR.

ABSTRACT Tomato is one of the most produced and consumed vegetables globally due to its nutritional benefits, sensory characteristics, and cultural importance. However, tomato fruit has a short shelf-life, which can be extended by postharvest techniques, but often at the expense of fruit quality, leading to consumer dissatisfaction. To address this challenge, we modified the upstream regulatory regions of Ripening inhibitor (RIN), a master regulator of tomato fruit ripening, utilizing a CRISPR/Cas9 multiplex system. This approach enabled the creation of a population of tomato fruit with mutations of varying severity, which could have far-reaching effects on the RIN-induced gene regulatory network in tomato fruit, leading to downstream changes in fruit traits. We have generated 264 first-generation (T 0 ) transgenic lines of RIN promoter mutants with diverse genetic lesions and RIN transcriptional levels. Our study revealed a non-linear relationship between promoter mutations and gene expression, highlighting the potential roles of certain types of mutations in regulating RIN transcription. Future work will focus on evaluating fruit traits from mutants with pronounced changes in RIN expression, as well as performing transcriptomic analysis to explore the mechanisms underlying fruit quality modifications due to genome editing.

ABSTRACT Alveolar rhabdomyosarcoma (aRMS) is a fusion-driven pediatric cancer with poor survival and limited therapeutic options. To uncover novel vulnerabilities, we employed complex-based analysis of the DepMap functional genomic data, identifying CDK8 as a dependency in aRMS. Both CDK8 knockout and pharmacologic inhibition impaired tumor cell growth and induced myogenic differentiation in vitro and in vivo . Compared to genetic loss, CDK8 inhibition induced more dynamic transcriptional changes. With a genome-scale CRISPR-Cas9 drug modifier screen, we determined that the maximal anti-tumor activity of the CDK8 inhibitor requires the presence of the Mediator kinase module and transcriptional cooperation with the SAGA complex. We further identified SIX4 as a key transcription factor mediating CDK8 inhibitor-induced transcriptional activation of myogenic differentiation genes and tumor cell proliferation. These findings suggest a distinct gain-of-function mechanism of the CDK8 inhibitor and establish a strong rationale for CDK8 inhibition as a differentiation-inducing therapeutic strategy in aRMS. STATEMENT OF SIGNIFICANCE We provide a framework for uncovering therapeutic targets by network-based analysis of functional genomic screens. We identify CDK8 as a druggable target in aRMS and determine that CDK8 inhibition drives myogenic differentiation and impairs tumor progression via a collaborative mechanism involving the Mediator kinase module, SAGA complex, and SIX4.

ABSTRACT Mitochondrial complex I (CI) deficiency represents a common biochemical pathophysiology underlying Leigh syndrome spectrum (LSS), manifesting with progressive multi-system dysfunction, lactic acidemia, and early mortality. To facilitate mechanistic studies and rigorous screening of therapeutic candidates for CI deficient LSS, we used CRISPR/Cas9 to generate an ndufs2 -/- 16 bp deletion zebrafish strain . ndufs2 -/- larvae exhibit markedly reduced survival, severe neuromuscular dysfunction including impaired swimming capacity, multiple morphologic malformations, reduced growth, hepatomegaly, uninflated swim bladder, yolk retention, small intestines, and small eyes and pupils with abnormal retinal ganglion cell layer. Transcriptome profiling of ndufs2 -/- larvae revealed dysregulation of the electron transport chain, TCA cycle, fatty acid beta-oxidation, and one-carbon metabolism. Similar transcriptomic profiles were observed in ndufs2 -/- missense mutant C. elegans ( gas-1(fc21) ) and two human CI-disease fibroblast cell lines stressed in galactose media. ndufs2 -/- zebrafish had 80% reduced CI enzyme activity. Unbiased metabolomic profiling showed increased lactate, TCA cycle intermediates, and acyl-carnitine species. One-carbon metabolism associated pathway alterations appear to contribute to CI disease pathophysiology, as folic acid treatment rescued the growth defect and hepatomegaly in ndufs2 -/- larvae. Overall, ndufs2 -/- zebrafish recapitulate severe CI deficiency, complex metabolic pathophysiology, and relevant LSS neuromuscular and survival phenotypes, enabling future translational studies of therapeutic candidates.