Selecting appropriate experimental systems is crucial in cancer research, where factors such as model relevance, cost, and resource availability guide decisions. A detailed understanding of the strengths and limitations of each model helps ensure their optimal use. We recently developed a human lung squamous cell carcinoma (LUSC) model using genetically engineered human bronchial epithelial cells (hBECs). These were studied through organotypic air-liquid interface (ALI) cultures and standard in vitro assays, including proliferation, invasion, and anchorage-independent growth. However, we did not evaluate whether the same mutant hBECs behaved similarly in vivo, or if in vivo models offered distinct advantages. To address this, we conducted a comparative phenotypic analysis of mutant hBECs derived from the same donor in both ALI cultures and xenografts in immunocompromised mice. Both models followed a similar oncogenic trajectory, involving squamous differentiation and activation of Nrf2 and PI3K/Akt pathways, characteristic of the classical LUSC subtype. However, some transcriptomic differences related to an increase in microtubule formation and cell motility in xenografts emerged. Additionally, xenograft gene expression profiles more closely matched classical LUSC tumours than ALI cultures. Importantly, we observed spontaneous squamous differentiation in the absence of SOX2 overexpression and detected selection for NOTCH1 mutations in specific in vivo mutants. Truncation of NOTCH1 promoted squamous differentiation and suppressed mucociliary features in ALI cultures, underscoring its role as a potential LUSC driver. In summary, mutant hBECs in vitro and in vivo showed largely consistent phenotypes, validating both systems. However, in vivo models can enable the unbiased discovery of new genetic LUSC driver genes. This highlights the complementary value of integrating both model types in LUSC research.

Bacterial defense systems present considerable barriers to both phage infection and plasmid transformation. These systems target mobile genetic elements, limiting the efficacy of bacteriophage-based therapies and restricting genetic engineering applications. Here, we employ a de-novo protein design approach to generate proteins that bind and inhibit bacterial defense systems. We show that our synthetically designed proteins block defense, and that phages engineered to encode the synthetic proteins can replicate in cells that express the respective defense system. We further demonstrate that a single phage could be engineered with multiple anti-defense proteins, yielding improved infectivity in bacterial strains carrying multiple defense systems. Finally, we show that plasmids that express synthetic anti-defense proteins can be introduced into bacteria that naturally restrict plasmid transformation. Our approach can broaden host ranges of therapeutic phages and can improve genetic engineering efficiency in strains that are typically difficult to transform.

Abstract BCR signal dependency is a hallmark of diffuse large B-cell lymphoma (DLBCL) and other B-cell lymphoid malignancies originating from germinal centers. Chronic-active BCR signaling, typical for the more aggressive activated B-cell subtype (ABC) of DLBCLs, is often attributed to activating mutations within the BCR signaling cascade and continuous stimulation of the BCR by autoantigens. In certain ABC-DLBCLs, the BCR forms an intracellular multiprotein supercomplex with TLR9 and MYD88, which generates signals from endolysosomes. However, it is not clear whether the internalization of BCR is required for sustained signaling, nor have the mechanisms responsible for BCR trafficking been defined. A detailed and mechanistic characterization of receptor trafficking and its consequences is crucial for elucidating new therapeutic targets. To address these questions, we developed DLBCL cell models with modified ovalbumin (OVA)-specific hypervariable regions (HVRs) in the BCRs using CRISPR-Cas9 technology. Modified BCRs were incapable of binding self-antigens, while still responding in a controlled fashion to stimulation with ovalbumin. Using these genetic models, we demonstrated that autoantigens drive a complex BCR-dependent signaling program and facilitate the assembly of the intracellular BCR-TLR9-IκB complex, promoting NFκB pathway activation. Furthermore, we showed that the binding of autoantigens to the BCR leads to the internalization of the BCR-autoantigen complex via clathrin-mediated endocytosis (CME). Using genetic models with inducible inhibition of this endocytic pathway, we found that BCR internalization is essential for the oncogenic activation of BCR-dependent signaling pathways and the formation of the BCR-TLR9-IκB complex in autoantigen-dependent ABC-DLBCL cells. Finally, CME inhibition with dynamin-2 antagonists, such as phenothiazine derivatives, reduces BCR signaling, cell viability, and synergizes with SYK and PI3Kδ inhibitors. Since phenothiazines have well-defined safety and pharmacokinetic profiles, our data provide a framework for the rational design of clinical trials employing these drugs in the autoantigen-dependent subset of DLBCL.

Duckweeds ( Lemnaceae ) have excellent potential for fundamental and applied research due to ease of cultivation, small size, and continuous fast clonal growth. However, their usage as model organisms and platforms for biotechnological applications is often limited by the lack of universal genetic manipulation methods necessary for transgene expression, gene editing, and other methods to modify gene expression. To identify suitable strains for genetic manipulation of the giant duckweed, Spirodela polyrhiza, we screened several genotypes for callus induction and regeneration and established genetic transformation. We have identified SP162 to be amenable to Agrobacterium -mediated transformation via tissue culture. The procedure is robust and reproducible across laboratories, allowing stable expression of different reporter genes and selectable markers, enabling CRISPR/Cas9-mediated genome editing. In addition, due to a weak small RNA-based silencing response, S. polyrhiza sustains prolonged periods of transgene activity in transient expression assays. To promote duckweed research and encourage the adoption of S. polyrhiza , we have made SP162 (ID#: 5676 ) and its genome publicly available and provide here detailed procedures for its cultivation and transformation. Furthermore, we created a web server to explore its genome, retrieve gene sequences, and implemented orthologous gene search and a gRNA design function for diverse CRISPR/Cas-based applications ( https://agxu.uni-mainz.de/SP162/ ).

Invasive lobular carcinoma (ILC) accounts for 15% of breast cancers yet lacks specific therapy because ILCs are underrepresented in clinical trials and preclinical models are lacking. We established intraductal xenograft models to test whether the clinical pan-lysyl-oxidase PXS-5505, now in phase trials for myelofibrosis can exploit the collagen-rich matrix dependency created by CDH1 loss. PXS-5505 remodels fibrillar collagen, and halts tumor expansion and metastatic seeding across ER+ and triple negative models without systemic toxicity. Genome-wide CRISPR screens reveal ITGAV and ITGB5 as synthetic lethal partners of CDH1 and LOX inhibition downregulates their expression together with MYC, NF-κB, and AP-1 transcriptional programmes. Collagen fibre density/alignment, and MYC/AP-1 gene signatures serve as pharmacodynamic readouts of drug activity. These data uncover a tractable ECM-integrin-MYC axis in ILC and nominate PXS-5505, alone or with endocrine therapy, for window of opportunity trials in this understudied breast cancer subtype. One Sentence Summary Targeting matrix remodelling in ILC inhibits ILC progression and alters multiple molecular endpoints, providing a translatable therapeutic strategy for this understudied subtype that requires better treatments.

Phosphorus (P) is an essential element limiting algal growth and productivity in aquatic ecosystems. Diatoms are important microalgae that thrive in nutrient-variable environments. Determining how diatoms perceive and respond to P availability is therefore crucial for understanding their ecological success. P-limited diatoms use a calcium (Ca 2+ )-dependent signalling pathway to sense and coordinate cellular responses to phosphate resupply. Despite the importance of Ca 2+ signalling for diatom environmental sensing, apparatus enabling Ca 2+ signal decoding is poorly understood. Here, we characterise the repertoire of an important group of Ca 2+ sensor proteins—Ca 2+ dependent protein kinases (CDPKs), in Phaeodactylum tricornutum . Several PtCDPKs are transcriptionally upregulated under P starvation. To determine whether PtCDPKs can coordinate P-starvation responses or act to transduce Ca 2+ signals induced by P resupply, we functionally characterised PtCDPK2. PtCDPK2 is highly expressed in P-limited cells and localises to the cell periphery, suggesting a role regulating plasma membrane processes. Further, PtCDPK2 is co-regulated with the transcriptional regulator of P-starvation responses, PtPSR1. PtCDPK2 expression is also coordinated with the induction of P-Ca 2+ signalling, which is driven by depletion of cellular P rather than external P exhaustion, or growth limitation. Ptcdpk2 mutants have significantly reduced photosynthetic efficiency and alkaline phosphatase activity under P starvation, but we do not find evidence for a direct role coordinating downstream responses to P resupply. These findings suggest PtCDPK2 is essential for regulating P-starvation physiology and reveals a role for Ca 2+ -signalling apparatus in promoting diatom tolerance in low P environments.

Tau aggregation is a pathological hallmark of a group of neurodegenerative diseases collectively termed tauopathies. While impaired proteostasis is known to drive the accumulation of abnormal proteins, the molecular factors influencing Tau aggregate clearance remain incompletely understood. In this study, we employed a cell-based Tau reporter assay combined with a lentivirus-based pooled CRISPR-Cas9 loss-of-function library to identify genes that regulate Tau aggregation. Genome-wide screening revealed that candidate genes were significantly enriched in categories related to mRNA metabolic processes and autophagy. Among them, we focused on the RNA-binding protein G3BP1, which is functionally associated with both processes. Detailed analyses showed that G3BP1 deficiency promoted the accumulation of Tau aggregates without affecting stress granule formation or autophagic flux. Instead, G3BP1 dysfunction resulted in impaired lysosomal homeostasis, as evidenced by reduced lysosomal abundance and acidification. Furthermore, lysosomal damage induced by LLOMe enhanced Tau aggregation, particularly in G3BP1-deficient cells. Conversely, pharmacological activation of TFEB by the curcumin analog C1 restored lysosomal function and suppressed Tau aggregate accumulation to wild-type levels. These findings highlight a role of G3BP1 in maintaining lysosomal homeostasis and promoting Tau clearance. Our results further suggest that therapeutic strategies aimed at enhancing lysosomal biogenesis, such as TFEB activation, may hold promise for the treatment of tauopathies.

The SRY-HMG-Box transcription factor SOX10 plays a critical role in neural crest development, but its function in epithelial tumorigenesis remains unclear. Here, we identify SOX10 as a key regulator of tumor-initiating activity in Neu-driven mammary cancers. Genetic ablation of Sox10 in the luminal compartment of MMTV-Neu (NIC) mice resulted in delayed but normal mammary gland development. Sox10 deletion conferred a dose-dependent delay in tumor onset, with a complete loss of tumor initiation in Sox10-deficient luminal cells. CRISPR/Cas9-mediated Sox10 inactivation in Neu-transformed tumor cells led to reduced 3D invasion and diminished self-renewal in mammosphere assays. Established Sox10-null cell lines exhibited markedly impaired growth in orthotopic transplant models and failed to colonize lung tissue following tail vein injection, suggesting a loss of tumor-initiating capacity. Transcriptomic profiling revealed that Sox10-deficiency in Neu+ tumor cells induces a luminal-to-basal/stem-like shift and the downregulation of several genes associated with genetic networks regulating stemness. Collectively, these findings demonstrate that Sox10 is required for a permissive cell progenitor state for Neu-driven tumor initiation and that it is critical to sustain the invasive and self-renewing traits that drive tumor progression and metastasis. Significance The Sox10 transcription factor is critical for mammary stem cell function and plasticity. Animal studies suggest that different subtypes of breast cancers arise from luminal epithelial cells and hypothesized the implication of luminal stem cells in breast cancer initiation. We show that Sox10 deletion in the luminal compartment of adult female mice abrogates tumor initiation. Sox10 inactivation impairs tumor cell growth, dissemination and reprograms luminal Neu+ tumor cells to a basal phenotype. Our data supports a model whereby luminal progenitors are required for tumor initiation, self-renewal and growth at distant sites. The findings provide a new therapeutic opportunity in the targeting of Sox10 networks to reduce the cancer stem cell content of mammary tumors.

ABSTRACT Metastatic breast cancer remains incurable, as many patients develop therapy resistance. Loss of ATM/p53 increases reliance on the ATR pathway, positioning ATR inhibitors (ATRi) as promising therapeutics. However, targeting a single pathway often leads to resistance due to tumour heterogeneity or alternative signalling mechanisms. Here, we combine chromatin-enrichment proteomics and phospho-proteomics with genome sequencing data in the ATRi-sensitive triple negative breast cancer (TNBC) cell line MDA-MB-453 to map adaptive responses to the ATR inhibitor AZD6738. We identify chromatin-associated activation of survival pathways, including AKT1, RPTOR (mTORC1), CDK4/5, and OGFR, alongside hyperphosphorylation of MKI67, SAFB2, and CHD4, indicating ATRi sensitivity. Complementary siRNA screening of DNA damage response (DDR) genes reveals that amplification of Fanconi anaemia (FA) pathway gene FANCE increases sensitivity to AZD6738. Analysis of breast cancer datasets highlights frequent FANCE amplification in metastatic patients, particularly in circulating tumour cells. Strikingly, pharmacological inhibition of the FA pathway (UBE2T/FANCL-IN-1) synergises with AZD6738. Together, our findings define adaptive resistance mechanisms to ATR inhibition and nominate FA pathway blockade as a rational combination strategy. Overall, our work provides fundamental insight into the complexity of DDR in metastatic breast cancer and offers a platform for mechanistic investigation, which can be exploited in cancer therapy.

ABSTRACT Despite the progress in understanding the circadian pacemaker, the specific mechanism by which it regulates sleep remains incompletely understood. We have previously demonstrated that a substantial number of genes are rhythmically expressed in the mushroom body (MB) Kenyon cells (KCs), including Pka-C1 , which encodes the catalytic subunit of protein kinase A (PKA). PKA-C1 plays a crucial role in promoting daytime wakefulness; however, the underlying mechanism remains elusive. Here, we show that the γ-lobe is the primary site of rhythmic Pka-C1 expression using a newly developed in vivo luciferase reporter. Through a combination of in silico analysis, CRISPR mutagenesis, and chromatin immunoprecipitation, we identify the transcription factor Onecut as a regulator of Pka-C1 transcriptional rhythms in γ-KCs. Furthermore, genetic trans-synaptic connectivity mapping and neuronal activity imaging reveal that the dorsal Lateral clock Neurons (LNds) provide inhibitory input to a subset of dopaminergic (DA) neurons in the protocerebral anterior medial (PAM) cluster, PAM-γ5, rhythmically modulating their activity. This, in turn, rhythmically activates MB γ-KCs via excitatory Dop1R signaling. Resulting γ-neuron activity rhythms drive Pka-C1 transcriptional rhythms through Onecut. Furthermore, these PKA-C1 rhythms reinforce neuronal activity rhythms, creating a feedback cycle between transcriptional and neural activity rhythms that promote daytime wakefulness. Our findings highlight the conserved role of DA in promoting wakefulness and offer mechanistic insights into its complex regulation. More generally, this work provides a mechanistic framework for how circadian rhythms are translated into neural activity to orchestrate complex behaviors like sleep.

ABSTRACT The glycan distribution on cells is governed by the stochastic activity of different families of enzymes that are together called ‘glycoEnzymes’. These include ~400 gene products or 2% of the proteome, that have recently been curated in an ontology called GlycoEnzOnto. With the goal of making this ontology more accessible to the larger biomedical and biotechnology community, we organized a web resource called GlycoEnzDB, presenting this enzyme classification both in terms of enzyme function and the pathways that they participate in. This information is linked to i) Figures from the “Essentials of Glycobiology” textbook, ii) General gene, enzyme and pathway data appearing in external databases, iii) Manual and generative-artificial intelligence (AI) based text describing the function and pathways regulated by these entities, iv) Single-cell expression data across cell lines, normal human cell-types and tissue, and v) CRISPR-knockout/activation/inactivation and Transcription factor activity predictions. Whereas these data are curated for human glycoEnzymes, the knowledge framework may be extended to other species also. The user–friendly web interface is accessible at www.virtualglycome.org/glycoenzdb .

Co-editing strategies have emerged as an approach to facilitate the selection of CRISPR/Cas-mediated mutants in a transgene-free manner: the gene of interest is edited together with a reporter gene, whose mutation can be selected visually or pharmacologically. In this work, we asses the impact of editing the well-used reporter Acetolactate synthase (ALS) on plant development and metabolome. We show that the desired mutation in ALS1 (P186S) conferring selectable herbicide resistance trait does not show significant impact on the plant morphology and physiology but that the additional mutations resulting from the same sgRNA can result in reduced vegetative vigor and altered metabolomic profiles in tomato.

Background Polycomb Repressive Complex 2 (PRC2) modulates chromatin accessibility and architecture to direct tissue-specific gene expression. PRC2 function is frequently altered in cancer by loss-of-function mutation or deletion, but the downstream effects on transcriptional regulation are incompletely understood. Results To gain insights into these mechanisms, we performed a holistic analysis of epigenomic and transcriptional changes in an isogenic model of acute myeloid leukemia (AML) with heterozygous EZH2 deletion that mimics reduced PRC2 function in patient leukemias. PRC2-depleted cells had diverse gene expression changes, including a bias towards more immature monocyte-lineage transcriptional signatures. PRC2 depletion also correlated with marked increases in chromatin accessibility genome-wide, with 10-45% increases in ATAC-seq peaks in EZH2+/− clones. These changes were accompanied by decreased H3K27me3 and increased H3K27ac levels in CUT+RUN assays that were incompletely linked to transcriptional activity. Despite these generalised changes, 3D chromatin architecture assessed by Hi-C was largely maintained, with H3K27me3 preferentially lost in regions with low DNA-DNA contact frequency. Surprisingly, some regions gained broad H3K27me3 domains at heavily compacted chromatin. We notably saw compartmentalisation changes upstream of the transcriptionally upregulated fetal hematopoiesis gene LIN28B in EZH2+/− cells, with corresponding activation of a LIN28B-specific transcriptional program, including upregulation of the CDK6 oncogene. These results correlated with EZH2+/− cell phenotype, including decreased cellular proliferation and increased resistance to CDK6 inhibitor palbociclib. Conclusions Our findings suggest that PRC2 depletion pleiotropically affects AML transcriptional regulation to directly impact cell phenotype and treatment responsiveness, which may partially explain the aggressive biology seen in these cases.

Cell signaling plays a critical role in regulating cellular state, yet uncovering regulators of signaling pathways and understanding their molecular consequences remains challenging. Here, we present an iterative experimental and computational framework to identify and characterize regulators of signaling proteins, using the mTOR marker phosphorylated RPS6 (pRPS6) as a case study. We present a customized workflow that uses the 10x Flex assay to jointly profile intracellular protein levels, transcriptomes, and CRISPR perturbations in single cells. We use this to generate a “glossary” dataset of paired protein–RNA measurements across targeted perturbations, which we leverage to train a predictive model of pRPS6 levels based solely on transcriptomic data. Applying this model to a genome-wide Perturb-seq dataset enables in silico screening for pRPS6 and nominates novel regulators of mTOR signaling. Experimental validation confirms these predictions and reveals mechanistic diversity among hits, including changes in signaling output driven by anabolic activity, cellular proliferation and multiple stress pathways. Our work demonstrates how integrated experimental and computational approaches provide a scalable framework for multimodal phenotyping and discovery.

Anti-bacteriophage systems like restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable identification of invaders. How Gabija, a highly prevalent nuclease-helicase anti-phage system, executes self- vs. non-self-discrimination remains unknown. Here, we propose that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting temperate phage D3 in Pseudomonas aeruginosa, Gabija functions early by preventing phage genome circularization in a non-abortive manner. Phage and plasmid DNA-end sensitivity to Gabija is licensed by a phage exonuclease and ssDNA-annealing protein. Unrelated F8 and JBD30 phages are sensitized to Gabija by Gam_Mu, a distinct DNA end-binding protein that antagonizes loading of the host repair complex RecBCD. Escape phages lacking these end-binding proteins become protected from Gabija by RecBCD activities, which also prevent Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes circularization of linear DNA devoid of RecBCD as a mechanism to identify foreign invaders.

ABSTRACT Synthetic biology enables the integration of sophisticated genetic programs into microorganisms, transforming them into potent vehicles for therapeutic applications. Engineering strategies for microorganisms are rapidly evolving, offering promising solutions for cancer therapy, microbiome modulation, digestive health support, and beyond. Developing novel tools to engineer safe, nonpathogenic microbial platforms is essential for advancing clinical therapies. In this work, we present an innovative engineering approach for the probiotic Escherichia coli Nissle (EcN), aimed at creating a safe and efficient chassis for the bioproduction of therapeutics. The EcN endogenous pM1 and pM2 plasmids were cured and re-engineered to introduce a CRISPR-Cas12 chromosome shredding device and a therapeutic-producing genetic circuit, thereby generating a nonproliferative therapeutic-delivery system. Next, we build an AI-based bioinformatic pipeline to predict Anticancer-Cell-Penetrating Peptides (ACCPP) candidates. As a proof-of-concept, a selected ACCPP was produced in the engineered EcN chromosome-shredded (CS) chassis. This strategy yields a robust and controllable platform for the safe production and delivery of therapeutics, paving the way for the future development of microbial therapies and their clinical applications. GRAPHICAL ABSTRACT

Mutations in the ciliary protein INPP5E, encoded by inositol polyphosphate-5-phosphatase E, can cause retinal degeneration as part of the ciliopathy Joubert Syndrome or non-syndromic retinitis pigmentosa (RP). INPP5E regulates the membrane makeup of the primary cilium, however its function in the specialized sensory photoreceptor cells of the human retina remain unclear. Here we utilize control and CRISPR/Cas9-generated INPP5E knock-out ( INPP5E KD ) human induced pluripotent stem cells (iPSCs) to generate retinal organoids (ROs). Through proteomic and immunofluorescence analysis we show that INPP5E plays an important role in early retinal development and photoreceptor progenitor cell differentiation. In mature ROs, INPP5E localizes to the connecting cilium of photoreceptors, and the loss of INPP5E leads to altered localization of ARL13B and Rhodopsin in mature photoreceptors. Furthermore, photoreceptor outer segment structure is affected, leading to elongated outer segment membranes in both cone and rod photoreceptors, suggesting an important role for INPP5E in photoreceptor outer segment membrane biogenesis. Together, these data underline the importance of INPP5E in retina development and photoreceptor structure and highlight the usability of retinal organoids to study protein function in a human context.

Cancer treatment remains challenging due to heterogeneous responses to immunotherapy across patients and tumor types. Innovative strategies are required to overcome immune evasion. We have identified the splicing factor SLU7 as essential for the survival of cancer cells from diverse origins. SLU7 knockdown induces R-loop accumulation, transcription-dependent genomic instability, DNA damage, and replication catastrophe, together with aberrant splicing and inhibition of nonsense-mediated mRNA decay (NMD) and/or DNA methylation. These alterations lead to the expression of neoantigens, interferon B1, endogenous retroviruses, and cancer-testis antigens, which would enhance tumor immunogenicity. Therefore, we propose SLU7 targeting as a dual-action therapy, combining direct tumor suppression with immune activation. Using various murine cancer models, including orthotopic liver tumors, and multiple molecular strategies—such as inducible CRISPR/Cas9, systemic delivery of chimeric siSLU7–nucleolin aptamers (APTASLU), and intratumoral injection of siSLU7-loaded nanoparticles—we show that distinct siSLU7 sequences and delivery platforms effectively inhibit tumor growth. Furthermore, SLU7 silencing synergizes with immune checkpoint inhibitors, amplifying anti-tumor responses. Our in vivo data demonstrate that SLU7 is a promising, versatile target for diverse cancers. Its multimodal mechanism offers potential to overcome tumor heterogeneity, reverse immune tolerance, and enhance immunotherapy efficacy.

Background: Collagen fibrils are the primary supporting scaffolds of vertebrate tissues, but the mechanism of assembly is unclear. Methods Here, using CRISPR-tagging of type I collagen, high-resolution light imaging, and SILAC labelling, we elucidated the cellular mechanism underlying the spatiotemporal assembly of collagen fibrils in cultured fibroblasts. Results Our findings reveal the multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiated the processes of collagen secretion and fibril assembly and identified the crucial involvement of endocytosis in the regulation of fibril formation. By employing Col1a1 knockout fibroblasts, we demonstrated the incorporation of exogenous collagen into the nucleation sites at the plasma membrane through these recycling mechanisms. Conclusions Our study sheds light on a complex and previously unidentified collagen assembly process and its regulation of health and disease. Mass spectrometry data were available via ProteomeXchange with the identifier PXD036794.

How genomic changes translate into organismal novelties is often confounded by the multi-layered nature of genome architecture and the long evolutionary timescales over which molecular changes accumulate. Coleoid cephalopods (squid, cuttlefish, and octopus) provide a unique system to study these processes due to a large-scale chromosomal rearrangement in the coleoid ancestor that resulted in highly modified karyotypes, followed by lineage-specific fusions, translocations, and repeat expansions. How these events have shaped gene regulatory patterns underlying the evolution of coleoid innovations, including their large and elaborately structured nervous systems, novel organs, and complex behaviours, remains poorly understood. To address this, we integrate Micro-C, RNA-seq, and ATAC-seq across multiple coleoid species, developmental stages, and tissues. We find that while topological compartments are broadly conserved, hundreds of chromatin loops are species- and context-specific, with distinct regulation signatures and dynamic expression profiles. CRISPR-Cas9 knockout of a putative regulatory sequence within a conserved region demonstrates the role of loops in neural development and the prevalence of long-range, inter-compartmental interactions. We propose that differential evolutionary constraints across the coleoid 3D genome allow macroevolutionary processes to shape genome topology in distinct ways, facilitating the emergence of novel regulatory entanglements and ultimately contributing to the evolution and maintenance of complex traits in coleoids.

Streptococcus suis is a major pig pathogen with zoonotic potential, posing an occupational risk to farmers and meat handlers. We characterised 110 S. suis strains from diseased pigs in Ireland (2005–2022) using whole-genome sequencing to investigate population structure and phage-host dynamics. We identified fifteen distinct serotypes, with serotypes 9 and 2 being the most dominant. In silico multi-locus sequence typing revealed high diversity within the collection, identifying several sequence types (STs), including 26 novel STs. Investigation of strain-level genomic clustering using PopPUNK against global S. suis genomes showed that the Irish isolates were phylogenetically dispersed across the broader global S. suis population rather than clustering in a single clonal group. The majority of Irish isolates fall within the ten established pathogenic lineages, including the highly virulent zoonotic lineage 1. A stable endemic clonal lineage was identified among Irish isolates, showing minimal genetic variation over a decade. Prophage analysis revealed novel viral taxa that were interspersed among known streptococcal phages, rather than clustering distinctly. Restriction-modification systems were the predominant anti-viral defence systems identified across genomes. CRISPR-Cas systems were present in limited strains but showed substantial targeting bias toward full-length prophages, indicating ongoing phage pressure. CRISPR spacers matched non- S. suis streptococcal phages, and phylogenomic analysis revealed that Vansinderenvirus phages clustered with S. suis rather than other S. thermophilus phages, suggesting evolutionary connections between phage lineages infecting different streptococci. This study presents the first comprehensive genomic characterisation of S. suis in Ireland, revealing a diverse population with significant implications for animal and human health.

Although haemoglobin variants are prevalent in low- and middle-income countries, the exact disease burden remains unknown due to a lack of diagnostic capacity. Traditionally, routine clinical haemoglobin variant diagnostics have relied on electrophoresis, which separates the haemoglobins based on size and charge differences. However, electrophoresis-based assays are limited in depth and coverage due to their inability to separate co-migrating variants at the pH employed. Importantly, for genetic counselling of pre-marital couples and prenatal screening of inherited haemoglobinopathy risk, molecular-based assays are required. Here, we leveraged the specificity and dual mismatch intolerance of en31FnCas9 to achieve differential identification of haemoglobin variants S, C, D, and E. Moreover, we demonstrate reliable differential detection of homozygous, heterozygous and compound states of haemoglobin variants due to en31FnCas9s intolerance to dual mismatch in the respective gRNA-target DNA complementarity. Furthermore, we coupled our en31FnCas9-based haemoglobin variant detection to the signal enhancement of recombinase polymerase amplification (RPA) to achieve reliable differential detection of haemoglobin variants from non-invasive saliva and urine samples in <60 minutes. Taken together, our study demonstrates the feasibility of functionalization of CRISPR-based diagnostics as a point-of-care technique towards achieving the democratisation of haemoglobin variant diagnosis in resource-limited settings.

Plasmodium species malaria parasites require invasion and replication within red blood cells to cause disease. Merozoite surface proteins (MSPs) are proposed to play a role in attachment of merozoites to RBCs and have long been considered as potential vaccine targets, but their functions during invasion are largely unknown. We applied targeted gene editing to investigate MSP4 and 5 function in P. falciparum , which causes most malaria mortality, and P. knowlesi , an in vitro culturable zoonotic species closely related to the widespread P. vivax . CRISPR-Cas9 gene-editing revealed that P. knowlesi MSP4 was not required for parasite growth in vitro . While P. knowlesi MSP5 could be functionally replaced by P. vivax MSP5, it was refractory to gene deletion. We confirmed the opposite for two different P. falciparum laboratory isolates where MSP4 is essential but MSP5 is dispensable. Attempts to select for reliance on the non-essential MSP (e.g. P. knowlesi MSP4 or P. falciparum MSP5) through long-term growth of inducible knock-out parasites, or via chimeric complementation of the essential MSP4 or 5 with the essential MSP from the other species, were unsuccessful. Live cell filming revealed a severe cell-entry defect with conditional knock-down of MSP5 protein expression in P. knowlesi . This study demonstrates differential importance of MSP4 and MSP5 during merozoite RBC invasion across human infecting malaria species, emphasises that vaccine candidates must be considered individually for the two most prominent human malarias and promotes MSP5 as a potential vaccine candidate for P. knowlesi and P. vivax . Significance For a malaria parasite to cause disease, the merozoite form of the lifecycle has to infect and replicate within human red blood cells. Proteins on the surface of the merozoite are considered as promising vaccine candidates, but the functions of these proteins are poorly understood. Here we demonstrate that two structurally similar merozoite surface proteins (MSP), MSP4 and MSP5, have differential importance between one human infecting malaria species compared to a second. The finding that MSP4 is essential for growth in one species, and MSP5 in the other, has implications for understanding invasion biology of malaria parasites and highlights that even structurally similar vaccine targets may need to be chosen specifically for each human infecting malaria species.

Succinate dehydrogenase (SDH)-deficient paraganglioma and pheochromocytoma (PPGL) are rare neuroendocrine tumors for which no effective targeted therapies currently exist. To uncover new potential therapeutic targets, we performed an unbiased CRISPR-Cas9 genetic screen in immortalized mouse chromaffin cells (imCCs) with and without Sdhb loss. Our screen identified genes that differentially affect cell proliferation in Sdhb -deficient versus normal imCCs. Notably, several subunits of the transcriptional Mediator complex emerged as potential tumor suppressors, as their loss selectively promoted growth of Sdhb -deficient cells. Most strikingly , we found that the neddylation pathway—required for ubiquitin-mediated selective protein degradation—plays a critical role in controlling cell growth and survival in Sdhb -deficient imCCs. Specifically, loss of the neddylation regulator Ube2m led to increased proliferation, while loss of Ube2f suppressed growth of Sdhb -deficient imCCs. Consequently, global neddylation inhibitor MLN4924 (Pevonedistat) and UBE2F-CRL5 axis inhibitor HA-9104 were shown to downregulate neddylation, suppressing UBE2F activity and selectively inhibiting growth of Sdhb -deficient imCCs. This unexpected result highlights the neddylation pathway as a promising druggable vulnerability in this cell culture model of SDH-deficient PPGL.

Efficient gene integration using RNA-guided endonucleases has not yet been achieved in the mitochondrial genome. Import of nucleic acids into mitochondria, a controversial feature, is essential for implementation of Cas9-mediated genome engineering of mitochondria. Import of short RNAs naturally occurs in mitochondria, and several putative import mechanisms and determinants have been proposed. However to date, import of gene-length RNA, required for gene integration in the mitochondrial genome, has never been described. The goal of this study was to devise and test experimental strategies to detect and improve the import of mRNA-sized RNA in mitochondria, using S. cerevisiae as model. A first fluorescence-based screening approach, relying on mitochondrial import of a fluorescent protein encoding mRNA was analyzed by fluorescence measurements, western blot and mRNA-FISH. Confounding results obtained with these different techniques made it difficult to unambiguously conclude on the occurrence of import of mRNA-sized RNAs into mitochondria. An adaptive laboratory evolution (ALE) approach, imposing a strong selection pressure for mRNA import to mitochondria, was then designed and tested to improve mitochondrial mRNA import. While the ALE approach did not improve mitochondrial mRNA import in the present study, it is a promising, unambiguous method for future studies testing different RNAs or mutants. The present study highlights remaining challenges in analytical techniques to identify RNA import to mitochondria, and introduces a novel application of ALE for studies on mitochondrial import of short and long RNA species.