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.

The E3 ubiquitin ligases RNF43 and ZNRF3 are key negative regulators of canonical WNT signaling, promoting turnover of the WNT receptors FRIZZLED and LRP5/6 at the plasma membrane. While their mechanism of action is well established, how RNF43/ZNRF3 themselves are regulated remains unclear. Here, we identify WNK kinases as novel upstream regulators of RNF43 through proximity labeling proteomics. Using gain- and loss-of-function approaches, we show that WNKs control RNF43 surface localization and thereby its ability to ubiquitinate and downregulate WNT receptors. Pharmacological inhibition of WNKs increases RNF43 membrane abundance and enhances WNT suppression, an effect abolished in RNF43/ZNRF3 double knockout cells and organoids. Mechanistically, WNK inhibition alters RNF43 trafficking and ubiquitination, revealing a role for WNKs in regulating its plasma membrane distribution. These findings define a new regulatory axis linking the pro-WNT activity of WNKs to RNF43/ZNRF3-mediated feedback inhibition. Targeting WNK now offers a novel therapeutic strategy to restore WNT pathway control in cancers with RSPO fusions or RNF43 mutations.

Ethylene glycol (EG), one of the main monomers of polyethylene terephthalate (PET), is an attractive target for microbial upcycling. Despite this interest, there is a limited number of described organisms that can efficiently metabolise EG. Here, we report the metabolic and biotechnological potential of Pseudomonas putida JM37 as a novel bacterial chassis for EG valorization. We show that JM37 efficiently grows on EG as the sole carbon and energy source, outperforming other Pseudomonas strains. Genome sequencing and directed mutagenesis revealed that genetic redundancies in the glyoxylate assimilation pathways underlie its robust EG metabolism. Beyond biomass generation, we demonstrated the biotechnological potential of JM37. This strain was able to accumulate medium-chain polyhydroxyalkanoates (mcl-PHAs), dominated by C10 monomers, directly from EG. Moreover, JM37 successfully expressed heterologous biosynthetic pathways, including a violacein biosynthetic operon and a PET-hydrolase which has been secreted actively into the extracellular medium. Together, our results support the use of P. putida JM37 as a versatile synthetic biology chassis for sustainable EG upcycling and as a promising platform for circular bioproduction.

Voltage-gated calcium channels communicate electrical signals in membranes of excitable cells into cellular responses like secretion of hormones and neurotransmitters, or the contraction of heart and skeletal muscle cells. Their activation properties are tuned to match their specific functions. Consequently, the different members of the calcium channel family activate over a wide range of voltages and with greatly differing speeds. The skeletal muscle Ca V 1.1 and the cardiac/neuronal Ca V 1.2 represent two structurally closely related channels with particularly slow and fast activation kinetics, respectively. Both channel paralogs associate with the auxiliary calcium channel subunit α 2 δ-1, which is a known regulator of activation properties. By expressing Ca V 1.1 and Ca V 1.2 with and without α 2 δ-1 in a new double-knockout muscle cell line, we demonstrate that α 2 δ-1 regulates activation kinetics of the two channels in opposite directions. Molecular dynamics simulation revealed a string of charged amino acids connecting α 2 δ-1 to the intrinsic speed-control mechanism of voltage-sensing domain I (VSD I) in Ca V 1.1. Charge-neutralizing mutations of any of these charged amino acids abolished the α 2 δ-1 modulation and accelerated current kinetics. Together, these results reveal the molecular mechanism by which the α 2 δ-1 subunit regulates the intrinsic speed-control mechanism in the VSD I of Ca V 1.1 calcium channels.

Current models of microRNA (miRNA) silencing posit that RNA-sequence rules are sufficient for canonical targeting of mRNAs by Argonaute 2 (AGO2), the central protein of the miRNA-induced silencing complex (miRISC). Using chimeric eCLIP in CRISPR-edited LIMD1 +/+ , LIMD1 +/− , and LIMD1 −/− human small airway epithelial cells (hSAECs), we reveal a transcriptome-wide dependency on LIMD1, an AGO2 adaptor, for effective miRNA targeting and repression. In LIMD1-deficient cells, miRNA loading is uncoupled from productive targeting: despite increased AGO2–miRNA interactions, complexes engage fewer transcripts and sites, reducing occupancy and more than halving both the breadth and depth of targeting. We also observe altered AGO2 positional footprints across targets in LIMD1-deficient cells. LIMD1 dependence is most pronounced at defined RNA contexts: weak (GC-poor) seed pairings, interactions involving evolutionarily young miRNAs or sites that nonetheless form thermodynamically stable duplexes, with these losses particularly enriched in coding sequences of rapidly evolving C 2 H 2 -zinc-finger genes. Even within canonical seed repertoires of individual AGO2–miRNAs, LIMD1 is most critical at poorly conserved sites, indicating that LIMD1 broadens miRNA regulation beyond ancient, deeply conserved targets. In culture, LIMD1 deficiency de-represses oncogenic proteins that, in vivo , inversely correlate with LIMD1 levels in normal lung and adenocarcinoma, where LIMD1 is characteristically reduced, and whose dysregulation predicts poor survival. Thus, LIMD1 emerges as a key determinant of miRISC architecture, targeting, and potency, challenging RNA-centric models of miRNA function and exemplifying how adaptor proteins diversify post-transcriptional regulation. Graphical Abstract LIMD1 defines the scope of miRNA-mediated targeting and repression AGO2-chimeric eCLIP in CRISPR-edited human small airway epithelial cells (hSAECs) shows LIMD1 is required for productive AGO2–miRNA engagement transcriptome-wide. LIMD1 deficiency reduces the AGO2–miRNA:targetome. Each AGO2–miRNA binds fewer targets, with lower occupancy per site and per transcript and fewer global silencing events. LIMD1 dependence is strongest for GC-poor seed-sites, less conserved miRNAs and sites, and thermodynamically stronger duplexes. Dosage-dependent effects of LIMD1 deficiency are broadly observed. Target-mRNA decay and translational repression are reduced in LIMD1-deficient hSAECs, increasing protein output. In vivo , LIMD1 LOH–associated deficiency is prevalent and typically clonal in NSCLC. LIMD1 expression inversely correlates with oncogene levels in normal lung and adenocarcinoma, and target dysregulation predicts poor survival.

Adaptive plant development is orchestrated, among others, by directional, intercellular transport of the phytohormone auxin. Self-organizing development, such as flexible vasculature formation, depends on the auxin canalization manifested by the gradual formation of auxin transport channels via the feedback between auxin signalling and transport. Herein, we identify MAKR6 as a key, novel component of this feedback. MAKR6 expression highly accumulates in vascular cells and is tightly regulated by auxin via the Aux/IAA-ARF-WRKY23 transcriptional network. MAKR6 is indispensable for auxin canalization-dependent processes including leaf venation, vasculature regeneration, and de nov o auxin channel formation from the local auxin sources. Mechanistically, MAKR6 directly interacts with the PIN1 auxin transporter to modulate its trafficking and polarization. MAKR6 also associates with and links two key receptor-like kinase (RLK) complexes involved in canalization, TMK1/4 and the CAMEL-CANAR. Together, our study establishes MAKR6 as a multifaceted regulator that couples transcriptional auxin signalling to PIN1 repolarization and coordinates multiple RLK-mediated signalling pathways in canalization. This provides mechanistic insights into auxin canalization and exemplifies a framework for exploring similar regulatory nodes in other developmental contexts.

DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is a powerful variant of single-molecule localization microscopy (SMLM) that overcomes the limitations of photobleaching, offers flexible fluorophore selection, and enables fine control of imaging parameters through tunable on- and off-binding kinetics. Its most distinctive feature is the capacity for multiplexing, which is achieved through a process known as Exchange-PAINT. This technique involves assigning orthogonal DNA strands to different targets within a sample and then sequentially adding and removing complementary imager strands that are specific to only one target at a time. However, manual Exchange-PAINT workflows are often inefficient, prone to drift and variability, and lack reproducibility. Here, we introduce a custom compressed-air-driven microfluidics system specifically designed for multiplexed SMLM. Featuring a stackable and modular design that is, in principle, not limited by the number of channels, the system ensures robust, reproducible, and material-efficient buffer exchange with minimal dead volume. It operates in both manual and automated modes and can be readily adapted to a wide range of commercial and custom microscopes, including wide-field, confocal, STED, and MINFLUX platforms. We demonstrate robust 5-plex Exchange-PAINT imaging in cancerous U2OS cells and, importantly, we establish multiplexed nanoscale imaging in fragile primary cardiomyocytes. These applications highlight the unique power of our platform to extend super-resolution multiplexing into physiologically relevant systems, thereby opening new avenues for biomedical research.

Summary Single-stranded DNA (ssDNA) gaps are a hallmark of BRCA-deficient cells, yet the mechanisms that safeguard these lesions remain unclear. Through a genome-wide CRISPR screen, we identified the RAD9A-HUS1-RAD1 (9-1-1) complex as essential for the survival of BRCA2-deficient cells through an ATR-independent mechanism. Loss of 9-1-1 in this context leads to the accumulation of PRIMPOL-dependent gaps that fail to undergo post-replicat ive repair, resulting in pathological expansion and increased DNA damage. This instability is driven by excessive EXO1-mediated degradation, as EXO1 depletion rescues the phenotype. We further demonstrate that the 9-1-1 complex is required for POLζ-dependent gap filling. We propose a model in which ssDNA gaps, when extended beyond a critical length, become inaccessible to TLS-mediated repair and are fully reliant on homologous recombination. These findings establish the 9-1-1 complex as key regulator of ssDNA gap stability and a promising therapeutic target in BRCA2-deficient cancers. Graphical Abstract

Chlamydia is an obligate intracellular bacterium that differentiates between infectious, non-dividing EBs and non-infectious, dividing RBs. Pathogenic Chlamydia species are unusual in lacking a peptidoglycan sacculus, yet they do synthesize a transient and localized peptidoglycan structure at the divisome of the RB during their polarized division process. Although several studies have described the components of the chlamydial divisome necessary to generate peptidoglycan at a specific site on the membrane, less is understood about how the peptidoglycan structure is degraded to allow for the daughter cell to form and the division process to complete. Amidases are key components of the cell wall in model system bacteria as they catalyze the degradation and remodeling of peptidoglycan, including in the division septum. Here, we characterized the cell division-associated amidase, AmiA_Ct, of Chlamydia trachomatis both in vitro and in vivo . Our in vitro data show that AmiA_Ct is a bona fide , metal-dependent amidase capable of cleaving peptidoglycan. AmiA_Ct complemented an E. coli amidase deficient strain and supported the growth and separation of daughter cells. To assess the function of AmiA_Ct in C. trachomatis , we generated a transformant strain carrying an inducible CRISPR interference system targeting the amiA gene. Knocking down expression of amiA resulted in altered bacterial morphology, a reduction in infectious EBs, and the accumulation of peptidoglycan in the organisms. These data indicate a critical function for AmiA_Ct in the unique cell division process of Chlamydia . Importance Peptidoglycan is an important structural cell wall polymer that serves to give bacteria their shape and resistance to changes in extracellular solute concentrations. For Chlamydia trachomatis , an obligate intracellular pathogen that divides within a host cell, peptidoglycan is only used for cell division and is not a component of its cell wall. In this study, we characterize the function of a chlamydial amidase that helps degrade peptidoglycan during cell division. We show a critical function for amidase activity in facilitating changes to the peptidoglycan structure during chlamydial cell division that support normal growth and development of this pathogenic bacterium.

Toxoplasma gondii, the causative agent of toxoplasmosis widespread in animals and humans, is an intracellular apicomplexan protozoan parasite infecting a variety of host cells. Gene editing using CRISPR-Cas9 has become a standard tool to investigate the molecular genetics of this interaction. With respect to gene knock-out (KO) studies, the general paradigm implies that the gene of interest is expressed in the wildtype and that only the gene of interest is affected by the knock-out. Consequently, the observed phenotype depends on presence or absence of genes of interest. To challenge this paradigm, we knocked out two open reading frames (ORFs) constitutively expressed in T. gondii ShSp1 tachyzoites, but not essential, namely ORF 297720 encoding a trehalose-6-phosphatase homolog and ORF 319730 encoding a You2 C2C2 zinc finger homolog. We analyzed the proteomes of tachyzoites isolated at a late stage of infection, of intracellular tachyzoites and of host cells at an early stage of infection. The intended KO proteins were present in the T. gondii Sp1 wildtype but absent in the KO clones. Moreover, besides differentially expressed (DE) proteins specific to each KO, 17 DE proteins common to both KOs were identified in isolated and 39 in intracellular tachyzoites. Moreover, 76 common DE proteins were identified in host cells. Network and enrichment analyses showed that these proteins were functionally related to antiviral defense mechanisms. These results indicate that the KO of a gene of interest may not only affect the expression of other genes of the target organism, in our case T. gondii, but also the gene expression of its host cells. Therefore, phenotypes of KO strains may not be causally related to the KO of a given gene.

Natural killer (NK) cells are emerging as a promising platform for engineered adoptive cell therapies. However, gene editing in NK cells remains challenging, and more effective strategies are needed. Here, we established a robust, feeder-free, and modular workflow for genome engineering in primary human NK cells, combining CRISPR/Cas9 with AAV6-mediated transgene delivery. Efficient site-specific transgene integration was achieved at various loci and can be coupled with concurrent disruption of the target locus in a single editing step. Furthermore, transgene expression was tunable according to the integration site and promoter. We applied this strategy to target a chimeric antigen receptor (CAR) transgene to a panel of inhibitory NK receptor loci, establishing a synergistic approach to enhance anti-tumor activity and facilitate the reliable comparison of CAR variants without expression bias. We identified TIGIT as an ideal locus that supports strong CAR expression and anti-tumor function. This genome engineering framework, which leverages multiple, complementary and precisely controlled genetic edits, can support the rational design of future NK-cell therapies tailored to overcome cell-intrinsic limitations and tumor-specific barriers.

The budding yeast Saccharomyces cerevisiae is a central model organism in genetics and synthetic biology, yet efficient multiplex genome editing remains difficult because many toolkits are restricted by limited plasmid selection markers and reduced efficiency when targeting multiple loci. In our previous work, we introduced a CRISPR-based system incorporating three nucleases with distinct PAM specificities but it was available only with the URA3 marker. Here we present LOBSTERS, an expanded modular vector series that retains the PAM-diverse nucleases (SpCas9, SaCas9, and enAsCas12a) while extending marker options to seven. This design enables the simultaneous use of multiple plasmids in one transformation, supporting scalable and flexible genome editing. Proof-of-concept experiments demonstrated efficient ADE2 and ADE3 deletions with colorimetric readouts, coordinated tagging of essential proteins (Cdc3 and Cse4) without compromising function, and recapitulation of three quantitative trait variants ( RME1, TAO3 , and MKT1 ) underlying sporulation efficiency. Together these results establish LOBSTERS as a robust and versatile platform for multiplex genome editing in S. cerevisiae . By enabling coordinated modification of essential proteins, genetic interactions, and quantitative trait variants, LOBSTERS provides a broadly applicable resource for functional cell biology and synthetic biology in yeast. Significance Statement LOBSTERS integrates three PAM-diverse nucleases with seven plasmid selection markers, overcoming the single-marker limitation of previous yeast genome editing toolkits. The system enables efficient simultaneous editing at multiple loci, demonstrated by functional tagging of essential proteins and recapitulation of quantitative trait variants. By broadening the editable space and lowering barriers to complex genotype construction, LOBSTERS provides a widely applicable resource for yeast cell biology and synthetic biology.

Cells have evolved organelle-specific responses to maintain protein homeostasis (proteostasis). During proteostatic stress, mitochondria downregulate translation and enhance protein folding, yet the underlying mechanisms remain poorly defined. Here, we employed cryo-electron tomography to observe the structural consequences of mitochondrial proteostatic stress within human cells. We detected protein aggregates within the mitochondrial matrix, accompanied by a marked remodeling of cristae architecture. Concomitantly, the number of mitochondrial ribosome complexes was significantly reduced. Mitochondrial Hsp60 (mHsp60), a key protein folding machine, underwent major conformational changes to favor complexes with its co-chaperone mHsp10. We visualized the interactions of mHsp60 with native substrate proteins, and determined in vitro mHsp60 cryo- EM structures enabling nucleotide state assignment of the in situ structures. These data converge on a model of the mHsp60 functional cycle and its essential role in mitochondrial proteostasis. More broadly, our findings reveal structural mechanisms governing mitochondrial protein biosynthesis and their remodeling under proteostatic stress.

Generation of arrayed genome-wide CRISPR libraries in a ready-to-transduce lentiviral format remains laborious, time-consuming, and costly. To address these limitations, the present study developed a fully automated lentivirus production and titration workflow using a Biomek i7 Hybrid automated workstation, integrated with multiple instruments and managed by SAMI EX software. The workflow produced and titrated viruses in 96 and 384-well plate formats, respectively. It employed reverse transfection and triplicate wells per lentivector to reduce variability and yielded an average of three viral particles in transduction unit (TU) per producing HEK293T cell. Titration was performed using U937-mCherry suspension cells, with the percentage of transduced cells converted from U937 (X%) to HEK293T (Y%) values via a linear regression equation (Y% = 4.3X% + 9.3%). The titer calculation was based on the initial seeding cell number, the converted percentage of HEK293T transduced cells, and virus input volume. The titration demonstrated strong reproducibility across LSRFortessa (BD) and Aurora (Cytek) flow cytometers (R 2 = 0.9). Among 1,760 unconcentrated virus preparations, median and mean titers reached approximately 1.2 x 10 6 TU/mL, with over 97% of samples exceeding the high-titer threshold of 2x10 5 TU/mL, thus demonstrating a robust, scalable, and cost-effective automation platform for high throughput arrayed lentiviral library production and titration.