The Icelandic mutation in the amyloid precursor protein (APP), APP A673T , has been identified in Icelandic and Scandinavian populations and is associated with a significantly lower risk of developing Alzheimer’s disease (AD). Although this mutation led to reduction in amyloid β-protein (Aβ) production, its effect on tau pathology is not well studied. We have crossed line 66 (L66) tau transgenic mice that overexpress the P301S aggregation-prone form of tau with C57Bl6/J mice expressing a single point mutation edited into the murine APP gene via CRISPR-Cas gene editing, termed APP A673T . We have performed ELISA, histopathological and behavioural analyses of heterozygous male/female L66 and L66xAPP A673T crosses at the age of 6 months to investigate the effect of the A673T mutation on tau brain pathology and behavioural deficits in these mice. Using immunohistochemistry, we found only a moderate, yet significant, reduction of mAb 7/51-reactive tau in prefrontal cortex for L66xAPP A673T compared to L66 mice. Quantification of tau in soluble/insoluble brain homogenate fractions by ELISA confirmed the lack of overt differences between genotypes, as did our extensive behavioural phenotyping using six different paradigms accessing motor function, olfaction, depression/apathy-like behaviour, as well as exploration and sociability. Therefore, the APP A673T mutation does not appear to modulate tau pathology or motor and neuropsychiatric behaviour in L66 tau transgenic mice.

ABSTRACT Accurate genome assemblies are critical for understanding small RNA-mediated genome defense. In animals, the PIWI-interacting RNA (piRNA) pathway protects genome integrity by silencing transposable elements. Studying how piRNAs are generated and how they guide heterochromatin formation requires complete reconstruction of genomic piRNA source loci and detailed transposon maps. Here, we present a high-quality de novo genome assembly of Drosophila melanogaster ovarian somatic cells (OSCs), a widely used cell line that recapitulates nuclear piRNA biology. The OSC genome differs substantially from the reference genome, with major differences in transposon content and piRNA cluster composition. Our assembly resolves the 700 kb flamenco locus, the primary piRNA cluster in OSCs, and provides a genome-wide transposon map. Using this resource, we characterize piRNA source loci, reveal how piRNA cluster composition determines transposon-derived piRNA profiles, and clarify the widespread impact of the nuclear piRNA pathway on heterochromatin. Finally, we provide an open platform for integrating user-generated datasets with the OSC genome, creating a community resource for studying transposon control and piRNA biology.

ABSTRACT T-cell based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy face substantial hurdles when confronting solid tumors such as ovarian cancer, where metabolic constraints in the tumor microenvironment limit T cell infiltration and function. In particular, T cells exposed to nutrient deprivation and hypoxia upregulate autophagy, a lysosomal degradation pathway that negatively regulates effector responses. Here, we used CRISPR-Cas9 to target a folate receptor alpha (αFR) CAR expression cassette into the locus of the essential autophagy gene ATG5 , thereby generating autophagy-deficient CAR-T cells in a single editing step. Targeted metabolite profiling revealed that deletion of ATG5 induced widespread metabolic reprogramming characterized by increased glucose and amino acid uptake. Functionally, ATG5 -knockout CAR-T cells maintained high cytolytic activity when assayed in patient-derived ascites in vitro , and exhibited superior and long-lasting tumor control against ovarian tumors in vivo . Taken together, our results suggest that deletion of ATG5 metabolically primes CAR-T cells for enhanced cytotoxicity in immune-suppressive conditions, thereby improving the therapeutic potential of αFR CAR-T cells for ovarian cancer immunotherapy.

Abstract Purpose Osteosarcoma (OS) is a prevalent primary malignant bone tumor that predominantly affects children, adolescents, and young adults. Proline-rich protein 11 (PRR11) is wellknown for its role in regulating cell cycle progression and promoting tumorigenesis. Nevertheless, the precise molecular mechanisms underlying PRR11-driven tumorigenesis in OS have yet to be elucidated. In the present study, we aimed to elucidate the role of PRR11 in OS and its underlying molecular mechanisms. Methods Genotype‒tissueexpression(GTEx) and The Cancer Genome Atlas (TCGA) data were analyzed for PRR11 expression (normal vs OS) and survival differences (low vs high expression). Immunohistochemistry(IHC) and western blotting(WB) were performed to examine the expression distribution of the PRR11 protein in OS tissues and cell lines. Three types of lentiviral vectors were used to establish stable 143B cell lines: (1) miRNA-based shRNA vectors, (2) Lenti-CRISPR-Cas9 vectors, and (3) overexpression vectors. RNA-seq analysis of the miRNA-based shRNAs. WB was used to elucidate the mechanisms by whichPRR11 affects DNA damage, DNA repair, the cell cycle, and the Hippo signaling pathway. Moreover, functional assays included colony formation, wound healing, and transwell assays in vitro and subcutaneous inoculation in vivo . Results This studyidentified PRR11 as a pivotal regulator that promotes OS cell migration, invasion, and proliferation in vitro and promotes OS subcutaneous inoculation. RNA-seq analysis revealed that PRR11 silencing regulates several signaling pathways, including the cell cycle, the DNA damage response, and DNA repair;subsequently, the detection of DNA damage/repair markers and cell cycle-related proteins further confirmed alterations in these signaling pathways. Subsequent flow cytometry experiments revealed that PRR11 could prolong the G0/G1 phase and shorten the G2/M phase. Conclusions PRR11 functions as an oncogene in OS, where the PRR11-Hippo axis drivestumor progression through a DNA damage-cell cycle coupling mechanism.

CRISPR-Cas9 is a gene editing tool used extensively in biological research that is now making3 its way into clinical therapies. With the first CRISPR therapy obtaining approval by the United4 States’ Food and Drug Administration (FDA) in late 2023, we look at clinical trials of emerging5 therapies involving CRISPR-Cas9, currently the most prevalent CRISPR-based tool in these6 trials. A CRISPR-based therapy is currently approved for treatment of both sickle-cell anemia and7 transfusion-dependent β-thalassemia but clinical trials for CRISPR-based therapeutics include a8 much broader range of targets. CRISPR-Cas9 is being explored to treat cancer, infectious disease,9 and more. This review highlights CRISPR-Cas9 clinical trials registered at clinicaltrials.gov as of10 12/31/2024.

Background In this study, we aim to develop a yearly updatable database that could predict chemotherapeutic drug resistance and overall survival probability in breast cancer patients. Existing drug sensitivity databases depend on correlation-based predictions. In our study, candidates involved in drug resistance are chosen based on cell line validation (overexpression or downregulation or inhibition of candidates) studies, curated manually. Method 28,773 mRNA expression signatures from 914 breast cancer patients were extracted from cProsite. 106 of these patients had clinical information and log2 fold change information required for this study. We categorized these patients into deceased and surviving groups from TCGA. To prepare a database that can predict drug resistance and overall survival, we included mRNAs that were over-expressed in at least 80% of the breast cancer patients and mRNAs over-expressed in deceased and surviving groups. In addition, we also reported breast cancer-associated drug resistance candidates which have been reported in cell-line based studies. The database matrix preparation involved an approximate of 15000 manual searches of cell validated studies. (750 candidates x 20 drugs). The database was validated using a publicly available breast cancer patient proteomics data. Results Our analysis identified a list of top priority candidates associated with multidrug resistance, categorized based on their resistance to >15 drugs, 5-15 drugs, and 2-4 drugs. Analysis of patient profiles in the database revealed that the number of proteins contributing to drug resistance was high in the poor prognosis category compared to the good prognosis category. Conclusions Our study highlights the probable gaps in breast cancer drug resistance research, as only a small subset of overexpressed mRNA candidates found in patients are studied in vitro or in vivo experiments focusing on drug resistance. We also identified candidates involved in multidrug resistance, whose role in drug resistance has not been studied in more than 15 drugs. After further validations, this will benefit the clinicians and upcoming CRISPR gene therapeutics.

Parkinson’s disease (PD) is a devastating neurodegenerative disorder primarily characterized by the progressive and unstoppable loss of dopaminergic (DA) neurons in the substantia nigra. We previously identified NATO3 (FERD3L), a conserved developmental transcription factor, as essential for maintaining DA neuron function during aging. Here, we show that AAV-mediated Nato3 gene transfer into the mouse substantia nigra prevents DA neuron degeneration in both MPTP-induced and α-synuclein (α-Syn) overexpression PD models. This neuroprotective effect is achieved by improving autophagic flux and ⍺ -Syn clearance. Furthermore, lentiviral-mediated NATO3 overexpression in human midbrain DA neurons, derived from induced pluripotent stem cells carrying the pathological ⍺ -Syn A53T mutation, effectively reversed key disease hallmarks. These include ⍺ -Syn accumulation, aberrant mitochondrial morphology, autophagic impairments, and compromised neurite structure. Collectively, these in vivo and in vitro findings highlight NATO3’s role in safeguarding DA neurons against pathological cellular events, positioning NATO3 as a therapeutic target for PD.

ABSTRACT Heterogeneous T cell states are critical in immune responses and have been explored by CRISPR-based and synthetic domain-swapped transcription factor (TF) screens, yielding novel insights and immunotherapeutics. However, a scalable strategy to map TFs in primary human T cells is lacking, which limits our understanding of the functions of critical TFs. We therefore adapted a transposon-based TF mapping strategy termed Calling Cards for primary human CD8 T cells, applying it to five key TFs with undefined binding sites in this cell type: TOX, TOX2, TCF7, SOX4, and RBPJ. To derive biological insights from these data, we developed an analytical framework to integrate TF binding with multi-omic sequencing data, revealing convergence of TOX and TCF7 binding at dynamic enhancers of memory CD8 T cells. We then identified TF co-bound gene programs related to memory and exhaustion states in addition to putative gene targets of known and unappreciated TF roles, including TOX binding at critical genes of both exhaustion and terminal effector memory differentiation. To further scale our TF analysis platform, we modified Calling Cards to create TFlex a method uniquely suited for multiplexed mapping of paralogous TFs. We applied TFlex to simultaneously map eight natural and domain-swapped TFs in primary human CD8 T cells, which demonstrated that domain-swapped TFs display emergent behavior in binding site selection and transcriptional effects on target genes that cannot be estimated as the sum of their constituent domains. Collectively, our data highlight the importance of scalable TF mapping in primary human T cells to elucidate TF function and the transcriptional regulation of cell states.

Abstract Background The genus Leuconostoc has significant importance in food fermentations and is increasingly evaluated for probiotic applications. Although Leuconostoc falkenbergense is a recently identified species, and its in-depth genomic characterization and probiotic attributes remain largely unexplored, particularly for strains derived from diverse natural sources such as traditional dairy food matrices. This study therefore aimed to isolate and characterize a L. falkenbergense strain (BSMRAU-M1L5) from naturally fermented traditional buffalo milk curd and performed whole-genome sequencing with detailed genomic analyses to evaluate its probiotic potential and safety profile for future applications. Results The draft genome of L. falkenbergense BSMRAU-M1L5 spans 1.78 Mb, assembled into 96 contigs, and encodes 1,844 genes. Phylogenetic and average nucleotide analyses with 21 other L. falkenbergense strains confirmed that BSMRAU-M1L5 is most closely related to strain C, previously isolated from yogurt in China. Pan-genome analysis revealed a substantial core gene set and 124 strain-specific genes in BSMRAU-M1L5, including 99 hypothetical genes, while the remainder were linked to metabolic versatility, survivability, and functional adaptation. Functional annotation revealed genes involved in carbohydrate utilization, vitamin and acetate biosynthesis, acid tolerance, antioxidant defense, stress response, adhesion, AI-2 signaling, exopolysaccharide production, and antimicrobial activity, highlighting its strong probiotic potential. The genome also harbors CRISPR-Cas systems, insertion sequence elements, and prophages, while lacking virulence and biogenic amine–related genes, confirming its genomic safety. Despite harboring three vancomycin resistance genes, the strain was susceptible to most tested antibiotics and exhibited γ-hemolysis, supporting its non-pathogenic nature and overall safety profile. Conclusion The genome of L. falkenbergense BSMRAU‑M1L5 encodes diverse metabolic and probiotic properties, supported by both genomic and phenotypic evidence of safety, positioning it as a promising candidate for use in fermented food production and broader biotechnological applications.

RNA localization and local translation mediate spatial and temporal control of polarized cells, including radial glial cells (RGCs) which produce and organize neurons and glia. Within RGCs, RNAs are transported long distances to basal endfeet, where they can undergo local translation. However, the subcellular composition of RGCs and function of local gene regulation remains largely unknown. Here, we discover that basal endfeet harbor a rich transcriptome including a Dynein component critical for subcellular RGC function. By purifying RGC compartments in vivo , we discover ∼3000 endfoot transcripts, including ∼800 highly enriched compared to cell bodies. Many endfoot-enriched transcripts exhibit conserved subcellular localization in neurons and glia and are associated with neurodevelopmental disease. We show that endfoot-enriched Dync1li2 regulates RGC basal morphology and subsequently interneuron organization. Finally, we develop LOCAL-KD, a CRISPR-Cas13 based method for subcellular mRNA knockdown in vivo . Leveraging this, we demonstrate that endfoot-localized Dync1li2 is critical for RGC morphology. Our study establishes experimental paradigms to understand RNA localization in the nervous system. Moreover, we discover RGCs have a vast subcellular transcriptome, revealing foundational insights into how RGCs control cortical development.

The emergence of multidrug-resistant (MDR) "superbugs" is a serious threat to world health because of the interdependence of environmental ecosystems, agriculture, and human medicine within the single health framework. Horizontal gene transfer (HGT), residue persistence, and excessive antibiotic use have accelerated the development of antimicrobial resistance (AMR) by 2050, it is predicted that AMR would be responsible for 8.22 million deaths. To evade conventional therapies, bacteria employ sophisticated resistance mechanisms such enzymatic inactivation (e.g., β-lactamases, carbapenemases), target alteration (e.g., PBP2a in MRSA), efflux pumps, biofilm formation, and reduced membrane permeability. In light of this dilemma, new strategies are needed to restore the efficacy of antibiotics and stop the spread of resistance. Advances in antibiotic adjuvants, like efflux pump blockers and β-lactamase inhibitors like avibactam, complement existing drugs to combat resistance. The arsenal against MDR pathogens is further diversified by phage therapy, CRISPR-Cas systems, anti-virulence inhibitors, combinatorial therapy and vaccinations. However, challenges persist, biofilm resilience, plasmid-borne anti-CRISPR defences, and ecological risks of gene-editing tools necessitate rigorous mitigation AI-driven diagnostics, metagenomics, and genomics together offer a revolutionary approach to AMR management and surveillance. To address the superbug epidemic & pandemic, the paper integrates the genetic, molecular, and ecological facets of AMR and highlights the vital need for international collaboration, sustainable practices, and One Health-aligned policies.

Intracellular photosymbiosis has evolved across life and forms the foundation of coral reef ecosystems. Using the sea anemone Aiptasia as a model, we generated a high-quality proteome of the symbiosome, the organelle that houses algal symbionts. This proteome revealed protein trafficking mechanisms and the types of biomolecules exchanged during symbiosis. Symbiosomal enrichment of lysosomal proteins, visualization of lysosomal fusion, along with reduced symbiosis following knockdown of lysosomal genes, supports its phagolysosomal identity and that extensive co-option of lysosomal proteins shapes the symbiosome. CRISPR/Cas9-induced mutations in the symbiosomal and lysosomal bicarbonate/sulfate transporter, SLC26A11, disrupted symbiosis in both Aiptasia and a reef-building coral. These findings support that anemones and corals independently evolved a carbon-concentrating and sulfate transport mechanism to fuel photosymbiosis by co-opting an orthologous lysosomal transporter.

ABSTRACT Cancer therapies are typically effective in subsets of patients, reflecting the molecular diversity of cancers and motivating the need for predictive biomarkers of response. Biomarker-guided therapy is increasingly useful in oncology, yet biomarker discovery remains complicated by the large number of molecular features that make it difficult to distinguish causal determinants from spurious associations. To address this challenge, we combined functional genomic screening, proteomics, and drug sensitivity profiling to discover response biomarkers for a number of therapies used in the treatment of Peripheral T-Cell Lymphomas (PTCL). First, we used genome-wide CRISPR-dCas9 interference screens in PTCL cells under drug treatment to identify a shortlist of genes whose knockdown directly increases or decreases drug sensitivity. Next, we profiled drug responses across a diverse panel of 30 PTCL cultures and, from the shortlist, identified genes whose protein abundance correlated with drug sensitivity. Genes detected by both approaches are causal determinants of drug response and correlates of drug response across the panel of lymphoma cultures, making them promising candidates for predictive biomarkers. Basal expression of the reduced folate carrier SLC19A1 was a strong predictor of pralatrexate sensitivity, consistent with its role as the primary transporter for pralatrexate uptake. Simulated clinical trials predicted that biomarker-guided patient selection could improve the power to detect significant benefit of adding pralatrexate to frontline chemotherapy in PTCL. These findings illustrate how the causal insights of functional genetic screens can augment correlative studies to identify biomarkers of drug response, and suggest the potential for precise use of pralatrexate for PTCL.

PIWI-interacting RNAs (piRNAs) are critical for transposon silencing and genome integrity, as well as gene expression regulation and antiviral immunity in metazoans, yet the molecular mechanisms governing their biogenesis remain incompletely understood. The participation of the splicing-associated process in piRNA biogenesis has been emphasized in multiple species, but the key factors and mechanisms remain elusive. Here, we identified SUGP1 (SURP and G-Patch Domain Containing 1) in BmE (a unique model cell system with a complete piRNA biogenesis pathway) as a key splicing factor that functions in piRNA biogenesis. Through CRISPR-Cas9-mediated gene knockdown in cultured cells combined with RNA-seq, small RNA-seq, and IP-mass spectrometry (IP-MS), our results reveal that SUGP1 deficiency disrupts piRNA accumulation, alters mature piRNA length distributions, and activates transposon expression. Immunofluorescence and Western blot (WB) analyses further demonstrate that SUGP1 interacts with Y-box protein (YBP), which is key regulators of RNA metabolism. Functional validation in Drosophila SUGP1-RNAi lines highlights evolutionary conserved and species-specific roles of SUGP1 in piRNA maturation. Collectively, our data uncover a dual role for silkworm SUGP1 in coordinating YBP-dependent piRNA biogenesis, thus elucidating a novel mechanistic framework for piRNA pathway regulation. Our work also underscores the silkworm as a unique model for studying non-canonical piRNA biogenesis mechanisms, with implications for treating transposon dysregulation-linked diseases. Author summary Disruption of piRNA synthesis leads to abnormal consequences such as transposon de-repression, posing a significant threat to genomic stability. It is therefore essential to in-depth analysis of the piRNA biosynthetic mechanism. Previous studies have highlighted the involvement of splicing-related processes in piRNA biosynthesis, yet key factors and mechanisms remain poorly understood. This study employed the silkworm cell system, which possesses a complete piRNA biosynthetic pathway. Utilizing CRISPR-Cas9-mediated gene knockout technology combined with RNA sequencing, small RNA sequencing, and immunoprecipitation-mass spectrometry (IP-MS), we discovered that SUGP1 deficiency disrupts piRNA accumulation, alters the length distribution of mature piRNAs, and activates transposon expression. Furthermore, immunofluorescence and Western blot analyses confirmed an interaction between SUGP1 and Y-box proteins (YBPs), key regulators of RNA metabolism. Functional validation using Drosophila SUGP1-RNAi lines revealed that SUGP1 plays dual roles in piRNA maturation. Our study reveals that the silkworm scissor-related factor SUGP1 has dual functions in coordinating YBP-dependent piRNA biosynthesis.

Cancer represents a complex adaptive system whose therapeutic recalcitrance is fundamentally driven by multidimensional heterogeneity. This heterogeneity manifests across genetic, epigenetic, metabolic, and immune axes, enabling tumors to evolve rapid and robust resistance mechanisms to monotherapies. While targeted therapies and immunotherapies have revolutionized oncology, their efficacy remains constrained by pre-existing and adaptive resistance in a majority of solid tumors. Herein, we propose a novel, integrated systems oncology framework designed to preemptively address this adaptive resilience. Our approach synthesizes four synergistic, clinically validated modalities: (1) Dynamic Immune Reprogramming (DIR) using high-fidelity CRISPR-Cas12b for in vivo checkpoint disruption and inducible CAR-T systems; (2) Metabolic Modulation via engineered extracellular vesicles (EVs) for mitochondrial transfer to reverse the Warburg effect; (3) AI-Driven Neoantigen Prediction employing ensemble machine learning models and federated learning to enable personalized, CRISPR-synthesized mRNA vaccines; and (4) Targeted Epigenetic Therapy utilizing lipid-coated mesoporous silica nanoparticles (LC-MSNs) for tumor-selective demethylation. We provide a rigorous technical elaboration of each pillar, supported by preclinical evidence, comparative analyses of enabling technologies (e.g., AAV9 vs. LNP delivery, autologous vs. allogeneic EVs), and mathematical models of clonal dynamics. The framework is critically evaluated within the context of translational hurdles, including immune-related adverse events, manufacturing scalability, and regulatory pathways. We present computational validation using TCGA data from 10,000 patients across 33 cancer types, molecular dynamics simulations of CRISPR-Cas12b systems demonstrating 2.3-fold improved specificity over SpCas9, and machine learning performance metrics showing our ensemble neoantigen prediction model achieves AUC = 0.91. Furthermore, we embed this scientific roadmap within a robust ethical and economic discourse, advocating for open-source platforms, equitable access models, and global partnerships. By synthesizing cutting-edge advances across molecular biology, bioengineering, and computational science, this manuscript serves as a comprehensive framework supported by computational evidence and data-driven validation. It outlines a pragmatic pathway for developing combinatorial therapies capable of constraining cancer's evolutionary capacity and achieving durable clinical responses across diverse and resource-variable settings. Methodological Innovation: The framework introduces several methodological innovations, including the application of federated learning for privacy-preserving neoantigen prediction across institutions, the development of tunable CAR-T systems with rapid on/off switching capabilities, and the creation of pH-responsive nanoplatforms for spatially-controlled epigenetic modulation. These technological advances are coupled with novel computational approaches, such as ensemble machine learning models that integrate multi-omic data streams to predict therapeutic resistance pathways before they emerge clinically. Clinical Implications: From a clinical perspective, this integrated approach promises to transform cancer from a terminal diagnosis to a manageable chronic condition across multiple solid tumor types. By simultaneously targeting multiple vulnerability axes, the framework aims to achieve synergistic therapeutic effects while minimizing the evolutionary escape routes that typically lead to treatment resistance. The modular design allows for adaptation to specific cancer subtypes and patient profiles, enabling truly personalized combination therapies that can be dynamically adjusted based on real-time monitoring of therapeutic response and resistance emergence. Global Health Impact: The framework embeds accessibility as a core design principle through open-source platforms, distributed manufacturing, and tiered pricing strategies. This ensures advanced cancer therapies can reach patients across economic spectra, addressing both scientific challenges and ethical imperatives for equitable benefit.

Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. Mass spectrometry-based proteomics has allowed proteome-scale quantification of hundreds of thousands of ubiquitination sites (ubi-sites), however the functional importance and regulatory roles of most ubi-sites remain undefined. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. We identified a core subset of ubi-sites under evolutionary constraint through alignment of ubiquitin proteomics data from six non-human species, and determined ultra-conserved ubi-sites recurring at regulatory hotspots within protein domains. Perturbation proteomics revealed that these highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize functional ubi-sites with roles in cellular signaling, we constructed a functional score for more than 100,000 ubi-sites by integrating evolutionary, proteomic, and structural features using machine learning. Our score identifies ubi-sites regulating diverse protein functions and rationalizes mechanisms of genetic disease. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying protein ubiquitination.

Although bacterial genomes encode numerous potential toxins, it is unclear how evolution drives the specificity of these important virulence factors. Using an insect CRISPR screen, we identified the transmembrane protein Attractin (ATRN) as the receptor for Nigritoxin (Ntx), a Vibrio toxin that causes seasonal shrimp pandemics. We found that Ntx’s effector “warhead” inhibits translation via a previously uncharacterized mechanism. Moreover, we show that two related toxins require ATRN for entry but possess unrelated effector domains. One has a Rho-GTPase AMPylation function and the other an actin-targeting/proteolysis function. Our findings reveal the mechanism of Ntx entry and toxicity and show that the ATRN-targeting domain can deliver disparate effector domains, strongly indicating that this class of exotoxins can evolve as modular proteins using a common entry domain.

Abstract Background Heterozygous mutations in the Glucocerebrosidase gene ( GBA1 ), which encodes the lysosomal enzyme β-glucocerebrosidase (GCase), are a genetic risk factor for Parkinson’s disease (PD), characterised by lysosomal dysfunction. The pathological effects of GBA1 mutations on PD, especially their influence on lysosomal function, mitophagy, and mitochondrial bioenergetics, remain unclear. Methods Fibroblasts and dopaminergic neurons, generated from induced pluripotent stem cells (iPSCs) derived from patients with GBA1-PD, were used in the study. Live-cell imaging was performed to assess lysosomal acidification, protease activity, mitochondrial membrane potential, and mitophagy. Mitochondrial cristae density and autophagic vesicles were examined using transmission electron microscopy. Oxygen consumption rate was measured by Seahorse assay. V-ATPase assembly was evaluated using FLIM-FRET, and pharmacological interventions included rapamycin and acidic nanoparticles. Statistical analyses involved unpaired t-tests, one-way ANOVA, and two-way ANOVA. Results GCase activity, lysosomal acidification, protease activity, mitophagy and mitochondrial bioenergetic function were all impaired. Mitochondria were fragmented, with reduced membrane potential and oxygen consumption. MTORC1 was constitutively phosphorylated and FLIM-FRET measurements confirmed impaired V-ATPase assembly, which was reversed following rapamycin treatment. Rapamycin and lysosome-specific acidic nanoparticles rescued lysosomal pH, restored mitophagy and mitochondrial membrane potential in GBA1 mutant dopaminergic neurons. Conclusions Our findings identify lysosomal acidification as the primary cause of impaired bioenergetic function and reduced mitophagy in GBA1-PD. MTORC1-mediated disruption of V-ATPase assembly drives these pathogenic processes. Pharmacological interventions that restore lysosomal pH—such as rapamycin or acidic nanoparticles—rescue both lysosomal and mitochondrial defects, offering a promising therapeutic approach for GBA1-PD.

Abstract Genome-wide sequence analysis has identified the SPI1 gene as a genetic risk factor for Alzheimer's disease (AD). SPI1 encodes the PU.1 protein, which plays a critical role in microglial development and immune responses, primarily studied in mouse models. However, no studies have yet reported the impact of the SPI1 gene on AD-related phenotypes in zebrafish. Therefore, this study utilized CRISPR/Cas9 gene editing to generate spi1a knockout zebrafish mutants, investigating the effects of spi1a loss-of-function on AD-associated phenotypes. The results showed that spi1a knockout led to reduced locomotor activity and increased brain cell apoptosis in larvae, while working memory, acetylcholinesterase (AChE) activity and Aβ1–42 levels remained unchanged. In contrast, adult spi1a knockout zebrafish exhibited significant cognitive decline, upregulated apoptosis-related genes, elevated AChE activity and increased Aβ1–42 accumulation. Transcriptomic analysis further revealed that spi1a knockout altered the expression of multiple AD-related genes and affected immune and inflammation-related signaling pathways. In conclusion, spi1a deficiency induced AD-like phenotypes in adult zebrafish. This study demonstrates the role of spi1a in modulating AD-related phenotypes in both larval and adult zebrafish, providing crucial insights into AD pathogenesis and establishing a valuable model for future high-throughput drug screening and therapeutic development.

The exponential growth of non-coding RNA research—with over 230,000 papers published since 2000—has created an urgent knowledge management crisis in molecular biology. Despite their crucial regulatory roles, microRNAs (miRNAs) face a significant curation bottleneck, with only 1,400 articles manually curated to the Gene Ontology (GO) knowledgebase over a decade. We present GOFlowLLM, an automated curation pipeline powered by reasoning-enabled Large Language Models (LLMs) that follows established GO curation flowcharts to extract and structure miRNA-mediated gene silencing data at scale. When evaluated on existing curation, GOFlowLLM selects the correct GO term in 90% of cases. Curators also agree with 95% of the system’s reasoning steps and 90% of the evidence selected. Applied to 6,996 previously uncurated articles, our system identified 2,538 new candidate GO annotations on 1,785 articles in just 58 hours—potentially doubling the available miRNA GO curation. Manual review of a subset of these annotations shows that curators agreed with the selected term in 87% of cases, the model’s reasoning in 92% of cases, and the extracted evidence in 93%. GOFlowLLM demonstrates how LLMs can significantly accelerate biocuration while maintaining high-quality standards by following expert-designed reasoning frameworks. The integration of reasoning traces in our system provides transparent justification for annotations that can be reviewed by human curators, addressing one of the key challenges in adopting AI for scientific curation, potentially transforming how we manage the growing corpus of scientific knowledge in molecular biology. GoFlowLLM is available on github: https://github.com/RNAcentral/GO_Flow_LLM .

Focal adhesions (FAs) are dynamic macromolecular assemblies that anchor the actin cytoskeleton to the extracellular matrix via integrin receptors, thereby regulating cell morphology and migration. Although FA maturation and organization have been extensively studied, it remains unclear how regulatory proteins influence the 3D architecture of FAs. Here, we show that loss of the vasodilator-stimulated phosphoprotein (VASP) impairs adhesion dynamics. We employed CRISPR/Cas9-mediated knockout of VASP and/or the mechanosensitive adaptor protein zyxin to investigate their respective roles in actin–adhesion coupling. Loss of VASP and zyxin correlates with altered FA morphology and impaired dynamics. Using cryo-electron tomography (cryo-ET), we resolved the polarity of individual actin filaments associated with FAs and identified a contractility-related actin layer enriched with tropomyosin. VASP and zyxin are required for the assembly of dense and aligned actin bundles with uniform polarity, oriented with their barbed ends towards the cell edge. In contrast, the tropomyosin-decorated dorsal actin layer remains unaffected by these perturbations. Our findings reveal distinct, layered architectures within FAs and underscore the cooperative role of VASP and zyxin in stabilizing the organization of actin filaments at functional adhesion sites.

Despite the central importance of epigenetic regulation in enabling adaptation and pathogenicity of malaria parasites, mechanisms controlling heterochromatin distribution in Plasmodium falciparum are poorly understood. Here, we identified P. falciparum Jumonji C domain-containing histone demethylase 1, PfJmjC1, as a key regulator of heterochromatin. Parasites lacking PfJmjC1 are viable in vitro but display slightly reduced multiplication rates compared to wild type parasites. Interestingly, PfJmjC1 depletion leads to major heterochromatin reorganization, involving de novo heterochromatin formation over non-essential, GC-rich euchromatic genes and reduction of heterochromatin at some chromosome ends, eventually altering the 3D genome architecture. Importantly, this heterochromatin reorganization results in the deregulation of cell surface antigen expression and failure of parasites to induce gametocyte production in response to nutrient deprivation. Collectively, our findings highlight the crucial role of PfJmjC1 in regulating heterochromatin distribution and life cycle progression in this deadly pathogen.

The development of CRISPR-Cas9 cleavage activity prediction tools hinges on data produced from high-throughput guide-target lentiviral library screens for different Cas9 variants. However, the majority of such tools remain limited to predictions for one or few Cas9 variants, making it difficult to quantify the effects of Cas9 residues on cleavage activity. To bridge the gap, we introduce 4 interpretable DeepEmbCas9 models for the cleavage activity prediction of 40 type II-A and II-C Cas9 variants — DeepEmbCas9, DeepEmbCas9-MVE, DeepEnsEmbCas9 naive, and DeepEnsEmbCas9 — leveraging protein and RNA language model embeddings to encode Cas9 and sgRNA, respectively. Among the 4 neural network models, DeepEnsEmbCas9 naive performed the best in both in-distribution and out-of-distribution settings, where DeepEnsEmbCas9 naive outperformed individual Cas9 cleavage activity prediction tools on 18 out of 51 and 17 out of 48 benchmark test sets, respectively, and performed comparably otherwise. Concerning uncertainty quantification, DeepEnsEmbCas9 yields quantile-calibrated uncertainty estimates while keeping a minimal performance drop compared to DeepEnsEmbCas9 naive. SHAP importance analysis on DeepEmbCas9 reaffirms the importance of Cas9-target PAM binding as a first step for Cas9 cleavage, and reveals the L2 linker and PLL-WED-PI as important Cas9 domains modulating DeepEmbCas9’s predicted activity change when introducing increased-fidelity and PAM-altering Cas9 mutations, respectively. Our findings demonstrate the usefulness of protein language model embeddings in uncertainty-aware Cas9 cleavage activity prediction. More generally, DeepEmbCas9 models serves as an initial step towards cleavage activity prediction modelling for the whole Cas9 protein family.

Clonal haematopoiesis (CH) arises from the expansion of hematopoietic stem cells (HSCs) carrying leukaemia-associated somatic mutations. CH is linked to pathological immune dysregulation and a greater risk of age-related inflammatory diseases. Yet, how CH mutations impact HSC differentiation into immune effector cells remains understudied. Here, we report a single-cell resolution functional and multi-omic investigation of HSC clonal and differentiation dynamics in individuals with DNMT3A-R882 CH. DNMT3A-R882 reshapes the clonal architecture of haematopoiesis towards an aged phylogenetic structure. Functionally, DNMT3A-R882 HSCs produce decreased monocytic output but more abundant and mature neutrophil progeny compared to WT HSCs in the same individual. Whereas DNMT3A-R882 myeloid progenitors display attenuated inflammatory transcriptional programmes, DNMT3A-R882 mature neutrophils acquire proinflammatory and immunomodulatory features typical of maladaptive immunity and CH co-morbidities. Our findings, validated in humanised mice, identify aberrant DNMT3A-R882 HSC-driven neutropoiesis as a key link between CH, immune dysregulation and risk of inflammatory disease.

Gene drive can control pathogen transmission or suppress vector populations by spreading drive alleles with super-Mendelian inheritance. CRISPR homing drive currently represents the most powerful type, and regulating Cas9 expression with specific promoters has been effective for improving drive performance. However, selecting these is often a major challenge. Here, we evaluated 35 Cas9 constructs driven by distinct promoters in different gene drive systems and identified associations between drive performance and single-cell RNA expression patterns of the promoter-associated genes. Our results indicate that higher drive conversion is significantly associated with elevated expression of the promoter-associated gene in the respective reproductive cells, but embryo resistance allele formation correlates with excessive female germline expression. For males, early germline expression produces superior performance. Thus, we find that optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window. In addition, found that in situ integrated rhino -Cas9 constructs significantly reduce somatic expression, underscoring the importance of genomic locus. On the basis of these results, we propose criteria for selecting promoters, providing a theoretical rationale and practical guidance for optimization of promoter elements in homing gene drive systems.