The myeloid oncogene TRIB2 is a key driver of acute myeloid leukaemia (AML) pathogenesis, promoting chemoresistance and blocking differentiation through ubiquitin-mediated degradation of the C/EBPα transcription factor. Despite its stable and sometimes elevated expression across AML subtypes, TRIB2 remains a clinically-untargeted vulnerability. Here, we present a comprehensive investigation into TRIB2 degradation mechanisms using multimodal approaches, including CRISPR knockout, mutational protein stability, small molecule TRIB2 engagement and evaluation of a novel targeted protein degrader (TRIB2-PROTAC). We identify Afatinib, a multi-ERBB covalent inhibitor, as a rapid inducer of TRIB2 degradation, triggering AML cell death via an ERBB-independent pathway. Importantly, TRIB2 degradation synergized with cytarabine, the frontline AML chemotherapy, amplifying therapeutic efficacy. Mapping of TRIB2 ubiquitination sites revealed Lys-63 as critical for its own proteolytic turnover, and a Lys to Arg degradation-resistant mutant (K all R) conferred enhanced chemoresistance and increased leukaemic engraftment in vivo . CRISPR-mediated TRIB2 knockout validated an essential role in AML cell survival. Consistently, the novel TRIB2-PROTAC (compound 5K) achieved robust TRIB2 degradation and AML cell killing at low micromolar concentrations. These findings establish TRIB2 as a compelling therapeutic target in AML and demonstrate that leveraging the ubiquitin-proteasome system to degrade TRIB2 offers a promising strategy to overcome chemoresistance. This work provides strong preclinical rationale for the development of TRIB2-targeting therapies in AML.

Plant immunity mediated by nucleotide-binding leucine-rich repeat (NLR) receptors often relies on canonical EDS1 / NDR1 signaling, but alternative mechanisms are emerging. We uncover a novel modular, noncanonical immune hub orchestrated by Rcr1 , a TIR-NLR (TNL) gene conferring clubroot resistance in Brassica napus against the root-infecting protist Plasmodiophora brassicae . Unlike typical TNLs, Rcr1 engages non-NLR partners in two separable modules: a CP1 (cysteine protease)–WRKY-based recognition module, likely monitoring a pathogen virulence target, and an AP (ankyrin-repeat protein)–ERF-based signaling module, driving jasmonic acid/ethylene-mediated defense. This architecture functions without detectable EDS1 / NDR1 involvement, challenging salicylic acid-dominant models of biotrophic immunity and expanding current views of how TNLs can be wired in plant defense. Using high-throughput interactor screening and CRISPR/Cas9 knockouts, we validate these modules, while heat-inducible gene excision reveals Rcr1 ’s critical early role (0–14 days post-inoculation). Together, our findings position Rcr1 as an exemplar of modular TNL architecture, suggesting that separable recognition and signaling branches may represent a broader principle of immune flexibility in plants. This study redefines TNL flexibility, offering a blueprint for breeding durable disease-resistant crops via modular immune engineering, with clubroot resistance as a model.

Only a fraction of bacterial genomes encode CRISPR-Cas systems but the selective causes of this variation are unexplained. How naturally virulent bacteriophages (phages) select for CRISPR immunity has rarely been tested experimentally. Here, we show against a panel of genetically and functionally diverse virulent phages that CRISPR immunity was not universally beneficial, and its fitness effect varied strongly between phages in predictable ways. In addition to mechanisms known to alter the effectiveness of CRISPR immunity, such as encoding a matching spacer or a protective nuclear shell, we show that the fitness effect of CRISPR immunity negatively correlated with the probability of evolving receptor-based resistance to the phage via spontaneous mutation. Supply of resistance mutations differed strongly between very closely related lipopolysaccharide-binding phages and was associated with variation at the C-terminus of the tail fibre protein altering residues involved in hydrogen bonding and the predicted binding site. Our results show that CRISPR immunity is more beneficial against virulent phages that are harder to evolve resistance to via receptor mutations, suggesting that virulent phage community composition and diversity will be important drivers of the prevalence of CRISPR immunity.

Abstract Tau is traditionally known for its role in microtubule stabilization, with its pathological aggregation central to tauopathies such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Recent evidence suggests that tau also plays important nuclear and nucleolar roles, yet the implications of tau pathology on nucleolar function remain poorly understood. Here, we show that tau localises to the nucleolus in both differentiated SH-SY5Y cells and iPSC-derived neurons, and accumulates upon expression of disease-associated MAPT mutations (P301S, S305N, and IVS 10 + 16). Using high-content imaging, we demonstrate that mutant tau expression leads to structural expansion of the nucleus and nucleolus, with upregulation of key markers from all three nucleolar sub-compartments, indicating increased in nucleolar activity. qPCR and nucleolar RNA-selective dye staining confirmed increased rDNA transcription and rRNA processing, suggesting that mutant tau drives elevated nucleolar biosynthetic output. This hyperactivation is accompanied by hallmarks of nucleolar stress and apoptosis, including p53 stabilisation, caspase 3/7 activation, and TUNEL positivity. These findings identify nucleolar dysfunction as a downstream consequence of mutant tau expression and highlight disruption of nucleolar homeostasis as a potential contributor to tau-mediated neurotoxicity in MAPT-linked FTD.

Influenza A virus (IAV) causes major economic losses to the poultry industry and poses a zoonotic threat to human health. Potential pandemic outbreaks are underpinned by the ability of the virus to jump from one species to another. Host-virus interactions can dictate the success of such events and while systematic studies have successfully mapped host virus interactions in human cells, few studies have been performed in relevant animal host cell lines. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens in chicken lung epithelial cells infected with either the human-adapted PR8 vaccine strain or the avian UDL 3:5 reassortant virus encoding PR8 HA, NA and M segments. Rather than selecting solely for cell survival, we used anti-M2 antibody staining and fluorescence-activated cell sorting to capture host factors influencing multiple stages of the IAV life cycle. Across both screens, we identified 104 genes required for efficient replication in chicken cells, including 16 with strong effects (log₂ fold change > 2). Comparative analysis with published human screens revealed 17 conserved host factors, 19 human-specific factors, and 42 chicken-specific factors, highlighting potential species-specific interactions. Top hits included genes involved in sialic acid biosynthesis and N-linked glycosylation— SLC35A1 , SLC35A2 , and the avian-specific influenza polymerase cofactor ANP32A . Functional validation demonstrated that MOGS , MGAT1 , DENR , DMXL1 , ENO1 , IPO9 , KLF6 , PTAR1 , and TSG101 contribute to multiple stages of the IAV life cycle. In particular, MOGS and MGAT1 were essential for N-glycan processing and modulated cell-surface sialic acid abundance, with strain- and species-specific effects. These findings define a genetic landscape of IAV dependency factors in chicken cells and suggest shared and species-specific host requirements that could impact cross-species transmission.

Genetic perturbations are one of the great strengths of the model organism Drosophila melanogaster , with approaches such as classical mutagenesis and RNA interference enabling a wealth of biological discoveries. A more recent approach for altering gene expression is CRISPR/Cas9-based mutagenesis, but as with any new tool, its use must be optimized. High expression of Cas9 has been shown to cause cytotoxicity in some cell types. Here, we show that Cas9 expression alone causes cytotoxicity in the dendritic arborization (da) neurons that are widely used to study neuronal development and regeneration. We then systematically evaluate alternative Cas9 transgenes designed to lower total Cas9 expression, called uCas9 transgenes. We show that expression of these uCas9 transgenes results in little to no cytotoxicity to da neurons. Lastly, we demonstrate the ability of uCas9 transgenes to effectively and specifically gene edit in da neuro ns. Thus, we expand the toolkit of genetic perturbations available to researchers working with Drosophila da neurons or other cell types suceptible to cytotoxicity due to high expression of Cas9.

ABSTRACT Nicotine is a plant-derived pyridine alkaloid with potent neurotoxic properties. A major pathway for detoxification of nicotine in mammals is via glucuronidation to produce nicotine N -glucuronide, but this process in insects remains poorly understood. Using mass spectrometry, we demonstrate that Drosophila melanogaster detoxifies nicotine through glycosylation, producing nicotine N -glycoside. Given that many new agrochemicals contain pyridine rings, we also investigated the metabolism of flonicamid and imidacloprid. We detected glycosylation of flonicamid, but not imidacloprid. A targeted RNAi screen across 21 UDP-glycosyltransferases ( Ugt s) identified Ugt35B1 as important for survival of nicotine exposure. CRISPR-based knockout of Ugt35B1 increases sensitivity to nicotine and flonicamid, but not to imidacloprid, nor to a structurally distinct neonicotinoid (thiamethoxam). Mass spectrometry of knockout and control flies confirms that Ugt35B1 glycosylates nicotine, its metabolite cotinine, and flonicamid. Together these findings establish Ugt35B1 as the principal UGT mediating nicotine detoxification in D. melanogaster , revealing a previously uncharacterized insect glycosylation pathway with potential implications for herbivory, insecticide detoxification and toxicology. Highlights - Drosophila detoxifies nicotine by glycosylation into nicotine N -glycoside. - A targeted RNAi screen identifies Ugt35B1 as critical for nicotine survival. - Ugt35B1 knockout sensitizes flies to nicotine and flonicamid, but not to imidacloprid or thiamethoxam. - First demonstration of an insect UGT mediating in vivo glycosylation of nicotine and cotinine.

The Fascin family of actin-bundling proteins organizes actin filaments (F-actin) into tightly packed bundles that drive dynamic membrane protrusions such as filopodia. In neurons, fascin has been thought to primarily function in axons, as previous studies reported its absence from dendritic filopodia and spines. Here, we demonstrate that fascin is both present and functionally important in dendritic compartments. Using optimized immunocytochemistry and CRISPR-based endogenous tagging of fascin1 in cultured hippocampal neurons, we show that fascin localizes to developing dendritic filopodia and is enriched in mature dendritic spines. Super-resolution imaging further reveals that fascin is organized into discrete nanoscale foci within spine heads, but not the spine neck. Finally, we show that CRISPR-mediated knockout of fascin1 in mature hippocampal neurons impairs synaptic potentiation, without affecting baseline excitatory synaptic transmission. Together, our findings uncover a previously overlooked aspect of actin organization in dendritic spines and establish fascin as a critical regulator of postsynaptic plasticity. Summary Statement The actin bundling protein fascin localizes to dendritic filopodia and spines, where it regulates activity-dependent synaptic plasticity.

ABSTRACT Chromosome segregation during anaphase occurs through two mechanistically distinct processes: anaphase A, in which chromosomes move toward spindle poles, and anaphase B, in which the anaphase spindle elongates through cortical astral microtubule pulling forces. Caenorhabditis elegans embryos have been thought to rely primarily on anaphase B, with little to no contribution from anaphase A. Here, we uncover a novel anaphase A mechanism in C. elegans embryos, driven by the kinesin-13 KLP-7 MCAK and opposed by the kinesin-12 KLP-18. We found that the extent of chromosome segregation during anaphase A is asymmetrically regulated by cell polarity cues and modulated by mechanical tension within the spindle, generated by opposing forces acting on chromosomes and spindle poles. Additionally, we found that the contribution of anaphase A to chromosome segregation increases progressively across early embryonic divisions. These findings uncover an unexpected role for anaphase A in early C. elegans development and reveal a KLP-7 MCAK -dependent mechanical coordination between anaphase A and anaphase B driven chromosome segregation. eTOC summary Dias Maia Henriques et al. uncover an anaphase A pathway, driven by the kinesin-13 KLP-7 and opposed by the kinesin-12 KLP-18, that contributes to chromosome segregation in early C. elegans embryos. Its activity is regulated by spindle tension, cell polarity cues, and progressively increases during early embryonic divisions.

In vertebrates, vitamin A (VA) is crucial for development, tissue homeostasis, vision, and immunity. Retinal, a form of VA, is produced via enzymatic cleavage of β-carotene by beta-carotene oxygenase 1 ( bco1 ) and bco1-like ( bco1l ). While bco1 is found across vertebrate taxa, bco1l is a paralog of bco1 that we discover to have evolved in the ray-finned fishes, the most abundant, speciose, and commercially important group of fishes. We investigated the function of bco1l in ray-finned Siamese fighting fish, commonly known as betta, an emerging model for genetics and development. Using CRISPR-Cas9 knockouts, we find that lack of bco1l results in reduced VA and elevated β-carotene in larvae, starting when animals have exhausted their yolk supply of retinal, followed by stunted growth and death during juvenile development. Exogenous retinoic acid rescues the mutation, demonstrating its deficiency causes these defects. bco1l is 4× more abundant than bco1 in the intestine. This, coupled with the inability of bco1 to sustain VA production in the bco1l mutant, indicates that bco1l is the primary enzyme for dietary carotenoid conversion into retinal. Our results show that VA production by bco1l is required for post-embryonic development, and that bco1l became essential after evolving via duplication of bco1 .

Homologous recombination (HR) is ubiquitous across evolution, driving adaptation by reshuffling standing genetic variation. Although bacteria lack meiotic recombination, HR extensively shapes their genomes. However, the mechanisms and ecological conditions sustaining frequent HR in bacteria remain unclear. Using Escherichia coli , we reveal how frequent recombination emerges from herd immunity to a generalized transducing phage. Herd immunity–established here via CRISPR immunity–maintains genetic polymorphism and enables stable host–phage coexistence, thereby promoting genome-wide gene flow and accelerating adaptation through recombination up to two orders of magnitude relative to de novo mutations. Notably, we show that recombination occurs in stationary phase and is mediated by RecG, which has been previously reported to be regulated by the stringent response – a bacterial reaction to nutrient deprivation and other stress conditions. Bacterial herd immunity thus fulfills an unexpected role of promoting adaptation by HR. This mechanism helps explain the enigmatic high rates of HR across bacterial populations, clarifies how bacteria adapt as resources wane, and suggests a broader evolutionary role for bacterial immune systems beyond individual defense.

ABSTRACT Plasmids are a foundational research reagent and a key material in biopharmaceutical manufacturing. Plasmids require a backbone for propagation in E. coli, which typically contains an antibiotic resistance gene alongside a replication origin. As plasmids increasingly enter clinical applications, concerns are raised on the safety risks of antibiotic resistance genes. Additionally, these protein-coding genes occupy long stretches of DNA that incur significant metabolic burdens on host cells, which negatively impacts plasmid manufacturability and functionality, leading to high production cost and compromised clinical efficacy. Here, we describe miniVec, a novel miniaturized plasmid backbone devoid of protein-coding sequence, and instead expresses a small RNA to provide constant selective pressure capable of sustaining high plasmid copy numbers in plain culture media devoid of antibiotics or other chemical additives. This simplifies large-scale fermentation and greatly increases plasmid yield. Notably, miniVec confers enhanced functionality in a variety of applications such as chemical transfection, electroporation, virus packaging, transposon-or CRISPR-mediated genome integration, and in vivo naked DNA transfection and vaccination, while exhibiting no detectable immunogenicity or toxicity. These advantages establish miniVec as the next-generation plasmid platform for clinical applications, featuring improved safety that aligns with regulatory expectations, enhanced manufacturability leading to much higher yield and dramatic cost reduction, and augmented functionality in diverse applications.

ABSTRACT Dravet syndrome (DS) is a severe childhood epilepsy caused by mutations of the sodium channel NaV1.1. These mutations are thought to compromise the ability of inhibitory interneurons to regulate network activity, leading to seizure events. However, standard treatments to restore inhibition have limited efficacy, suggesting the existence of additional pathological mechanisms. Here, we use hiPSC-derived neuronal networks containing both excitatory and inhibitory neurons to show that excessive bursting activity in DS cultures is driven by excitatory neurons. This rise in bursting frequency is caused by the increased expression of HCN1 “pacemaker” channels in excitatory neurons, and bursting activity can be normalised using a channel blocker. With this work, we propose a new pathophysiological mechanism in DS and identify HCN1 as a novel therapeutic target.

ABSTRACT Neuronal ceroid lipofuscinosis type 2 (CLN2) is a rare, fatal paediatric neurodegenerative genetic disease that causes progressive psychomotor decline, epilepsy, speech impairment, vision loss, and premature death by late childhood or early adolescence. It is caused by mutations in the TPP1 gene, which encodes the tripeptidyl peptidase 1 enzyme. To date, enzyme replacement therapy (ERT) with recombinant human TPP1 is the only approved treatment available and it does not appear to have a curative effect on disease progression or rescuing complex motor functions. This underscores the importance of developing more effective treatment strategies for CLN2 disease. In the quest for an alternative therapy, we here seek to utilise CRISPR based prime editing (PE) to correct one of the most frequent TPP1 mutations of c.622 C>T (p. Arg208Ter). In cultured mammalian HEK293a cells we screened 16 different PE and pegRNA combinations, achieving on-target editing efficiencies up to 27.36 %. To enable efficient in vivo gene editing, we packaged the most promising PE and pegRNA combination using engineered virus-like particles (eVLPs) which resulted in 12.6 % on-target editing. Given the robust on-target editing and reduced risk of bystander editing, PE is deemed suitable for in vivo testing in the pre-clinical models for CLN2 disease. Overall, our findings establish the proof of concept for CRISPR-based prime editing to correct pathogenic human TPP1 nonsense mutation of c.622 C>T (p. R208X) and provide a basis for further investigations of PE as a genetic treatment for CLN2 disease.

Abstract Aurora Kinase A (AurA) is an essential mitotic kinase and therapeutic target in cancer. Most protein kinase inhibitors target the conserved ATP-binding pocket, often resulting in poor selectivity and off-target effects. Here, we identify and characterise small synthetic protein binders, Adhirons, as allosteric inhibitors of AurA. Using ‘phage display, we isolated Adhiron reagents that bind a previously uncharacterised site on the αG-helix of the kinase C-lobe. Structural and biochemical analyses revealed that the Adhiron inhibited AurA by modulating the activation loop via this cryptic site, which we designate the T-pocket. In cells, Adhiron expression mimics the effects of small molecule inhibitors of AurA on substrate and auto-phosphorylation, while sparing Aurora kinase B and without impairing TPX2-mediated localisation of AurA to the mitotic spindle. The AurA-inhibitory Adhirons demonstrate remarkable selectivity, potency and affinity, a highly sought-after combination of properties for kinase inhibition facilitating their use as tractable research tools for probing AurA function and as pharmacophore templates for structure-based drug design. Finally, these reagents illustrate a generalisable strategy for targeting allosteric sites across the Kinome. *Jack P Roberts & James Holder contributed equally to this work.

Summary Abiraterone acetate serves as the first-line therapeutic agent for prostate cancer (PCa) treatment. However, drug resistance frequently emerges. Employing a genome-wide CRISPR/Cas9 library screening strategy, we identified 523 long non-coding RNAs (lncRNAs) and 2183 protein-coding genes associated with abiraterone resistance. Notably, a pair of sense-antisense genes, BIRC6-AS1 / BIRC6 , was demonstrated to be a pivotal driver for abiraterone resistance. BIRC6-AS1 depletion led to a reduction in both the mRNA and protein levels of BIRC6. Moreover, depletion of either BIRC6-AS1 or BIRC6 enhanced the chemosensitivity of PCa cells to abiraterone both in vitro and in vivo settings. Further investigation revealed that BIRC6-AS1 stabilized the mRNA of BIRC6 through interaction with ILF2. Diminishing either BIRC6-AS1 or BIRC6 predominantly suppressed non-homologous end joining (NHEJ) repair activity, resulting in the disassembly of 53BP1 foci at DNA damage sites and an increased accumulation of DNA damage in PCa cells induced by abiraterone. Mechanistically, BIRC6 interacted with A20 and facilitated the K48-linked ubiquitination and subsequent degradation of A20 at the K337 residue. Additionally, A20 knockdown effectively reversed the abiraterone sensitivity induced by BIRC6-AS1 depletion. Collectively, we conducted a comprehensive screen to identify lncRNAs and protein-coding genes associated with abiraterone resistance and proposed that targeting BIRC6-AS1 /BIRC6 axis represents a promising strategy to overcome abiraterone resistance in prostate cancer.

The success of plants on land has been enabled by mutualistic intracellular associations with microbes for 450 million years (Delaux and Schornack 2021). Because of their intracellular nature, the establishment of these interactions requires tight regulation by the host plants. In particular, three genes – SYMRK, CCaMK and CYCLOPS – form the core of an ancestral common symbiosis pathway (CSP) for intracellular symbioses, and are conserved since the most recent common ancestor of land plants (Radhakrishnan et al. 2020; Delaux et al. 2015; Wang et al. 2010; Parniske 2008). Here, we describe EPP1 as a fourth gene committed to the CSP. Among land plants, EPP1 is conserved only in species able to associate with at least one type of intracellular symbiont. We found that loss-of-function epp1 mutants or EPP1 knock-down lines in four clades of land plants – legumes, Solanaceae, monocots and bryophytes – are all impaired in their ability to associate with arbuscular mycorrhizal fungi. We discovered that the plasma membrane-localized receptor-like SYMRK phosphorylates EPP1 on a conserved serine residue and that this phosphorylation is essential for symbiosis. Using a gain-of-function approach, we demonstrate that EPP1 is upstream of the nuclear kinase CCaMK. We propose that EPP1 is an ancestral component of the essential pathway that has regulated plant symbiosis for half a billion years.

Indoor vertical farming (VF) offers several practical advantages for the cultivation of plant protein bio-factories, including plant uniformity, product consistency, water/nutrient recycling and production cycles on a year-round basis. Much progress has been achieved in recent years toward the development of innovative systems for artificial lighting, automated irrigation, plant handling, environment control and space use optimization in VF systems. Here, we used a CRISPR-Cas9 gene editing approach to generate mutant lines of transient protein expression host Nicotiana benthamiana presenting a compact, space-efficient phenotype compared to the so-called LAB strain commonly used for protein production. Our strategy consisted of altering apical dominance by suppressing the biosynthesis of strigolactone, a negative regulator of axillary bud outgrowth-promoting cytokinins. Strigolactone-depleted lines were generated by knocking-down the expression of either Carotenoid cleavage dioxygenase 7 (CCD7) or Carotenoid cleavage dioxygenase 8 (CCD8), two key enzymes of the metabolic pathway leading to strigolactone synthesis. Knocking-down the genes of either enzyme had no impact on the overall growth rate of the plant but drastically influenced its leaf proteome, auxin/cytokinin ratio and overall architecture. More specifically, the ΔCCD mutants exhibited altered glycolytic and malate-processing enzyme fluxes driving the production of pyruvate and cytokinins in leaf tissue, an axillary growth-oriented development pattern and, most importantly, a spatial footprint reduced by 45% to 50% compared to the LAB strain. Most importantly, recombinant protein yields per plant were maintained in the mutant lines, as here illustrated for the model protein GFP and for rituximab, a chimeric monoclonal antibody of confirmed clinical value in humans. Our data demonstrate the usefulness of ΔCCD7 and ΔCCD8 knockout leading to strigolactone depletion for the generation of compact, space-efficient N. benthamiana lines well suited to VF systems intended for biopharmaceutical production.

Emerging evidence that circulating levels of key metabolic intermediates are sensed by a range of G-Protein Coupled receptors (GPCRs) is providing critical new insights into the control of systemic metabolic homeostasis, and how disturbances in such sensing may contribute to metabolic disease. The hydroxycarboxylic acid receptors for lactate (HCAR1), β-hydroxybutyrate (HCAR2), and octanoate (HCAR3) are encoded by three closely homologous GPCR genes co-located in a region where common genetic variation has been reportedly associated with lipid levels and body fat distribution. By resolving sequence homology in this region, we were able to refine this signal to a coding variant (R311C) in HCAR2. Using corrected genotypes from ∼500K participants from UK Biobank and direct genotyping of four other studies, we found that carriage of the HCAR2 p.R311C variant was significantly associated with type 2 diabetes risk, reduced gynoid fat mass, increased waist-hip ratio, higher circulating triglycerides, glucose and alanine aminotransferase levels, lower levels of HDL cholesterol and adiponectin and impaired suppression of circulating levels of non-esterified fatty acids after oral glucose. Adipose tissue explants from mice engineered to express the equivalent mutation variant (p.R308C) in the mouse ortholog showed increased lipolytic activity, basally and after β-hydroxybutyrate (BHB) treatment. In vivo, the mice were insulin resistant and had increased liver fat and impaired post-prandial suppression of NEFAs. The variant alters an amino acid located in the intracellular C-terminal tail of HCAR2, increasing recruitment of β-arrestin and resulting in enhanced internalisation and reduced cell surface expression. In conclusion, a common variant in the human ketone body receptor results in impaired control of adipocyte lipolysis and adversely impacts systemic lipid and glucose metabolism. These findings highlight the importance of anti-lipolytic ketone body signalling in adipocytes for the maintenance of metabolic health Graphical Abstract

Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods using transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we developed CRISPR-Cas9 engineered HEK293T cell lines with stable integration of mCherry-C1C2 fusion proteins at the AAVS1 locus for continuous production of surface-modified EVs. The engineered cell lines demonstrated significantly higher surface display efficiency compared to transient transfection, with reduced batch-to-batch variability. EVs maintained native characteristics including size distribution (120-130 nm) and marker expression while showing efficient cellular uptake. The platform maintained consistent production of uniformly modified EVs with stable transgene expression over at least 25 passages (~3 months), eliminating the need for repeated transfections and reducing batch-to-batch variability inherent to transient expression systems.

Hepatocellular carcinoma (HCC) is a malignant tumor that has been associated with dysbiosis of the gut microbiota. However, how the gut microbiota plays an oncogenic role in HCC remains largely unknown. Here, we show that Enterococcus faecalis (E. faecalis) is highly enriched in liver tumor tissues and is positively correlated with pathogenesis of HCC. E. faecalis promotes liver cancer cell proliferation, protein translation, cell migration and tumorigenesis. Mechanistically, we found that EF-derived extracellular vesicles (EF-EVs) deliver EF-Obg GTPase to activate host mTOR, thereby promoting liver cancer progression. Intriguingly, the EF-Obg protein exerts its influence on the mTOR pathway via a Ras-like G domain involved in GTP binding. EF-Obg exhibits a striking homology with the G1 site of the G domain of Rheb, a key positive regulator of mTOR. Obg gene is critical for E. faecalis to activate mTOR to promote hepatocarcinogenesis based on an engineered obg knockdown strain by CRISPR interference experiment. Clinically, abundant EF-Obg protein expression is correlated with enhanced activation of mTOR, leading to poor overall survival in HCC patients. Significantly, treatment of mTOR inhibitor Everolimus confers effectiveness in EF-colonized liver cancer orthotopic model, suggesting that Everolimus therapeutic approach can be effective for liver cancer patients with enriched E. faecalis . Taken together, we provide mechanistic and functional evidence to verify a direct causal relationship between tumor-resident E. faecalis enrichment and liver carcinogenesis, revealing that EF-Obg functions as a previously unidentified cross-kingdom activator of mTOR to promote liver tumorigenesis.

Single-cell perturbation sequencing technologies (e.g., Perturb-seq, CROP-seq), which integrate CRISPR-based gene editing with single-cell transcriptome profiling, have revolutionized the analysis of transcriptomic changes induced by genetic perturbations at single-cell resolution. These technologies serve as a powerful tool for identifying key genes that inhibit tumor growth or reverse cancer cell phenotypes. However, they face two major challenges: data explosion with high experimental costs, and data complexity characterized by high dimensionality, noise, sparsity, and heterogeneity. To address these challenges, we developed the single-cell Rank-based Genetic Perturbation predictor (scRGP), the first deep learning framework leveraging gene expression rank-order information for this task. scRGP demonstrates superior performance in terms of robustness, cross-cell-line perturbation prediction, and high-throughput screening. Specifically, scRGP achieves an approximately 10-16 percentage points improvement in Pearson correlation coefficient (PCC) over state-of-the-art methods (e.g., GEARS and scFoundation) for single- and double-gene perturbation predictions, while also extending prediction capability to triple-gene perturbations. Furthermore, it outperforms these methods by approximately 5-9 percentage points in cross-cell-line predictions. These advancements promise to shift the paradigm of single-cell perturbation studies from experiment-driven to computation-driven approaches, providing new support for functional genomics and precision medicine.

CRISPR–Cas systems are central to prokaryotic adaptive immunity, widely harnessed for biotechnology. Yet, their vast and uncharacterized diversity, especially non-canonical variants, impedes full exploitation. Here we present BioPrinCRISPR, a class-agnostic computational framework leveraging gene co-conservation, protein domain co-occurrence, and embedding similarity to identify and characterize CRISPR–Cas systems across prokaryotic genomes. Applying BioPrinCRISPR to over one million bacterial genomes, we uncovered extensive canonical and uncharacterized systems, revealing a rich landscape of atypical Cas proteins and novel domain architectures. Notably, we identified recurrent fusion proteins with unique enzymatic combinations, suggesting roles in regulatory control or nucleic acid remodeling. Experimental validation of two divergent Cas13an-like effectors demonstrated RNA knockdown capacity in human cells, confirming our framework’s predictive power. These findings expand the functional repertoire of CRISPR-associated proteins and highlight unexplored modes of microbial immunity. BioPrinCRISPR thus stands as a powerful tool for comprehensively mapping CRISPR–Cas diversity, offering new insights into prokaryotic defense and facilitating discovery of novel candidates for next-generation genome engineering. An accompanying interactive web platform was also developed to facilitate data exploration.

Interleukin (IL)-1α is a pro-inflammatory member of the IL-1 cytokine superfamily and is important for inflammatory responses to infection and injury. Unlike pro-IL-1β, pro-IL-1α is mainly localised to the nucleus upon expression. This is mediated by a nuclear localisation sequence (NLS) responsible for its importin-dependent transport into the nucleus. This nuclear localisation and the presence of histone acetyl transferase (HAT)-binding domains within the pro-domain suggest a role of this cytokine in gene transcription regulation. In addition, nuclear trafficking of pro-IL-1α is proposed to regulate its secretion. To-date, studies on the nuclear role of pro-IL-1α have used overexpression systems. Here, we generated a mouse where the endogenous Il1a gene was edited with CRISPR to disrupt the NLS (mNLS). Using an in vitro approach with murine macrophages we found that this NLS mutation did not affect pro-IL-1α RNA expression levels in response to LPS but increased its protein expression levels. Moreover, we found that the transcriptional signature induced by LPS was not altered between WT and mNLS macrophages. Release of IL-1α in response to different stimuli such as ionomycin was not negatively impacted by disrupted nuclear localisation, although higher levels of IL-1α release were detected, potentially due to increased levels of pro-IL-1α. Inflammatory responses in an in vivo model of peritonitis and an influenza infection model were comparable between WT and mNLS mice. Thus, we have established a mouse model in which pro-IL-1α nuclear localisation is disrupted, although future research is required to reveal the importance of this nuclear localisation for IL-1α function.

Abstract In most solid tumors, hypoxia is a critical physical attribute that reprograms malignant cells into a highly metastatic state. Specifically, hypoxia is a well-established inducer of cellular plasticity, which is associated with treatment resistance and metastasis. Furthermore, hypoxia exacerbates chromosomal instability (CIN), a hallmark of cancer that can be initiated by the loss of Trp53 and a key contributor to metastasis. Despite this, the mechanisms by which malignant cells concurrently co-opt these elements of hypoxic adaptation to promote metastasis remains unknown. Here we report that hypoxia promotes metastasis by suppressing the JmjC-containing histone lysine demethylase Kdm8. CRISPR/Cas9-mediated targeting of Kdm8 in a Kras;Trp53-driven mouse model of pancreatic ductal adenocarcinoma robustly rewires the malignant cell transcriptomic programs, leading to a profound loss of the epithelial morphology and widespread metastatic disease. Mechanistically, Kdm8 suppression in normoxia recapitulates major aspects of the global epigenetic changes and the transcriptomic rewiring induced by hypoxia. Moreover, Kdm8 deficiency leads to mitotic defects, increased micronuclei formation, Kras copy number gains, and enhanced CIN. Of note, disruption of Kdm8’s demethylase function phenocopies the effects of Kdm8 loss, whereas expression of hypermorphic Kdm8 variants that are resistant to hypoxic suppression reduces metastasis beyond the levels achieved by the wildtype counterpart. Through the suppression of Kdm8 demethylase activity, hypoxia unleashes a potent metastatic program by simultaneously advancing cellular plasticity and CIN.