Extracellular vesicles (EVs) are lipid-delineated nanoparticles that are produced by most cell types. EVs contain complex molecular cargoes that can have useful therapeutic or vaccine immunological effects. Cell-free gene expression systems can be used to produce membrane proteins in vitro , that can co-localise and integrate with exogenously added EVs. To advance this type of cell-free EV engineering we manufactured, isolated and characterised HEK293 cell EVs. These EVs were successfully cell-free engineered with several CD63-based membrane fusion proteins. In our most optimal conditions, up to 4.83 ×10 11 /ml of HEK293 EVs were successfully cell-free engineered with a fusion membrane protein incorporating CD63 I-shaped membrane-insertion topology transmembrane helix 3 (CD63ITM3) and monomeric green lantern (mGL). Finally, we also demonstrated that nano flow cytometry is a powerful tool for assessing cell-free EV engineering efficiency. In the future cell-free EV engineering could help accelerate future EV discoveries and the development of EV translational applications. Graphical abstract Technology readiness Cell-free systems are a well-established part of the engineering biology toolkit. Indeed, several applications are already at TRL9 stage, including commercially available cell-free protein synthesis kits, drug screening assays, field-tested medical biosensors, as well as therapeutic antibody manufacturing that has now reached cell-free reaction volume scales of up to 4500 L. However, cell-free extracellular vesicle (EV) engineering is currently at TRL3 / 4 stage. Whilst our study helps to further advance cell-free EV engineering, key challenges remain. To accelerate technology readiness, improvements in scalable EV isolation methods, increased eukaryotic cell-free protein synthesis yields, and assay automation will likely speed up cell-free EV engineering workflows. Furthermore, artificial intelligence-guided design workflows could be used to rapidly create community accessible libraries of EV membrane protein scaffolds, therapeutic, cell targeting and other modular elements for use in EV engineering studies. Finally, the development of EV potency assays for functional assessment of cell-free engineered EVs are needed to progress prototyped designs towards clinical translation. If these challenges are addressed, we envision that cell-free EV engineering will become an important approach in EV biological research and clinical applications. Highlights Cell-free extracellular vesicle (EV) engineering could be utilised to rapidly prototype and test novel biotechnological, vaccine, or therapeutic EVs for foundational or translational applications. Our data highlight some of the impacts that hollow fibre-based cell culture, EV isolation methods and different cell-free engineering approaches have on cell-free EV engineering workflows. We demonstrate several characterisation assays and technologies, including nanoflow cytometry, that can be used to assess cell-free EV engineering efficiency.

The recent advent of long-read whole genome sequencing has enabled us to create an accurate telomere-to-telomere reference genome, construct pangenome graphs, and compile precise catalogs of genomic structural variations (SVs). These comprehensive SV repositories provide an excellent opportunity to explore the role of SVs in genotype-phenotype associations and examine the mechanisms by which SVs are introduced through double-strand break (DSB) repair. Here, we employed comprehensive SV catalogs identified through various short- and long-read whole genome sequencing efforts to infer the underlying mechanisms of SV introduction based on their genomic and epigenomic profiles. Our findings indicate that high local DNA methylation and DNA shape-related features, such as low variations in propeller twist, support the origins of homology-driven SVs. Subsequently, we utilized an active-learning-based unsupervised clustering approach, revealing that the homology-dependent SVs show greater evidence of retaining ancestral recombination patterns compared to their homology-independent counterparts. Finally, our comparison of inherited and de novo SVs from healthy populations and rare disease cohorts showed distinct upstream H3K27me3 levels in de novo SVs from individuals with ultra-rare disorders. These findings highlight genome-wide characteristics that may influence the choice of repair mechanisms linked to heritable SV origins.

RNA is at the forefront of therapeutics and gene editing technologies. Yet, RNA synthesis remains expensive and low-yield. Consequently, most oligo manufacturers abstain from synthesizing RNA oligos longer than 60-mers. Solid-phase synthesis is the current standard production method but is often fraught with low coupling yields for canonical nucleotides and even poorer coupling for modifications. This results in high levels of byproducts such as truncations and RNA infidelity. Existing analytical methods can only provide quality control metrics such as RNA length distribution or limited composition information for short oligos. Here, we developed a standard quality control metric using Oxford Nanopore direct RNA sequencing to obtain direct insight into RNA length distribution, sequence, and presence of RNA modification sites. Our pipeline identifies error-prone regions and truncation sites that occur during synthesis. Furthermore, problematic steps in the synthesis are identified and repaired. We show that our platform can produce and assess CRISPR guide RNAs with high-fidelity and higher cleavage activity, and further, that modifications can be reliably detected. We envision that our tool will serve as an integral method for quality control pipelines that assess the integrity and accuracy of synthetic RNAs and guide the improved synthesis and yield of synthesized RNAs. Graphical Abstract

Prochlorococcus is the most numerically abundant photosynthetic organism in the oceans and plays a role in global carbon cycling. Despite its ecological significance and the availability of over a thousand assembled genomes, progress in understanding gene function has been limited by the lack of genetic tools. Here, we report a reproducible electroporation-based protocol to introduce replicative plasmids into two strains of Prochlorococcus representing different ecotypes: MIT9313 (low-light adapted) and MED4 (high-light adapted). Using plasmids carrying a spectinomycin resistance cassette, we achieved transformation in ~33% of MED4 and ~10% of MIT9313 attempts, with greatest success when electroporating cells in late exponential phase. Transformed cells stably retained plasmids and expressed resistance genes, demonstrating functional uptake and gene expression. We also delivered a modified 13 kb plasmid carrying a CRISPR-Cpf1 system into MED4. While no targeted edits were observed, cpf1 and specR were expressed, indicating successful delivery of large constructs and active transcription. These findings represent a key step toward genetic manipulation of Prochlorococcus , enabling future optimization of gene editing approaches and deeper functional analysis of its vast and largely uncharacterized pangenome.

Background Climate warming promotes the expansion of insect pests. Among the inducible defense responses activated by attacked plants, Kunitz trypsin protease inhibitors (KTIs) play an outstanding role. KTIs affect food digestion and thereby control the fitness of herbivorous insects. Poplars contain an expanded family of KTIs, whose distinct intrinsic functions are under investigation. Here, we set out to identify KTIs with anti-herbivore activity and assessed the potential growth trade-off incurred by high KTI expression levels. Results We identified 28 KTIs in the haploid genome of Populus x canescens , 21 of them were responsive to herbivory. The greatest induction was observed for KTI_400, KTI_600 and KTI_0882 ( P. trichocarpa orthologues Potri.019G124400, Potri.019G124600, Potri.019G088200), whereas a moderate response was found for KTI_53200 (Potri017G153200 orthologue), a protein mainly localized in the xylem sap. Mechanical wounding and methyl-jasmonate resulted in fast and strong induction of KTI_400 and KTI_600 and moderate or lacking responses in KTI_0882 and KTI_53200 . Increased KTI expression levels were associated with upregulation of ALLENE OXIDE SYNTHASE (AOS), whereas exposure to compounds eliciting ethylene or salicylic acid signaling did not affect KTI s. We generated stable CRISPR/Cas12a-mediated knock-out and p35S -mediated overexpression lines of KTI_400, KTI_600 and KTI_53200 in Populus x canescens. Among the wildtype and transgenic lines, only kti_400+kti_600 double knock-out lines produced greater biomass. Larvae of Helicoverpa armigera, a pest expanding in Europe due to a warmer climate, were allowed to feed on wildtype and transgenic poplar lines. Transgenic poplars overexpressing KTI_400 or KTI_600 resulted in reduced and their double knock-out lines in increased weight gain of the larvae. In contrast, overexpressing or knock-out lines of KTI_53200 had no effect on larval weight gain compared with controls. Conclusion KTI_400 and KTI_600 are potent, natural in-planta anti-herbivorous agents. Their expression is associated with larval growth reductions. Modulation of KTI_53200 levels had no direct effects on the fitness of leaf-feeding H. armigera or on plant growth. This study sheds light on the potential application of KTI in plant defenses and biocontrol against herbivores in trees and presents new options to investigate growth-defense theories.

Heterochromatin Protein 1α (HP1α) is a fundamental component of constitutive heterochromatin, forming subnuclear condensates whose regulation and function remain poorly understood. Here, we present an image-based CRISPR screen targeting nuclear factors that identifies splicing as a pivotal pathway regulating HP1α condensates. We discovered that unspliced intronic RNA modulates HP1α condensates by interacting co-transcriptionally with HP1α. By modulating the intron content, RNA processing restricts HP1α-RNA interactions at chromatin, thus enabling heterochromatin organization. Disruption of HP1α condensates due to enhanced interactions with unspliced RNA leads to loss of heterochromatin and the activation of stress response protective genes. We propose that RNA is a central component of heterochromatin that modulates HP1α condensates, and that RNA processing enzymes act as a surveillance mechanism for condensates by dynamically regulating the network of multi-valent interactions between RNA and chromatin factors. This model underscores the crosstalk between chromatin organization, transcription, and RNA processing, potentially governing broader nuclear functions.

Phosphoinositide-specific phospholipase C (PI-PLC) is a signaling enzyme that hydrolyzes membrane phosphoinositide to generate lipid- and lipid-derived second messengers. In plants, PI-PLCs have been implicated in various physiological processes, including immunity. Tomato SlPLC2 was previously shown to be implicated in susceptibility to the necrotrophic fungus Botrytis cinerea . However, whether this observation extends to pathogens with different lifestyles and evolutionary origins, remains unknown. Here, we investigated SlPLC2 function during infection with Phytophthora infestans , a globally devastating oomycete and causal agent of potato and tomato late blight. CRISPR-Cas9 knockout SlPLC2 plants showed significantly reduced disease symptoms, lower pathogen biomass, and decreased sporangia production, indicating impaired colonization. Upon infection, SlPLC2 knockout lines displayed attenuated expression of salicylic acid (SA)- and jasmonic acid (JA)-responsive genes suggesting disrupted hormone signaling. Consistent with PLC role during plant defense, early immune responses such as hydrogen peroxide accumulation and callose deposition were reduced in knockout plants. At the cellular level, these plants allow fewer infection vesicles formation by P. infestans , accompanied by an increase in expression of a biotrophy-associated effector gene PiAvrblb2 , suggesting impaired establishment of infection. In Nicotiana benthamiana SlPLC2-GFP localizes predominantly at the plasma membrane. Upon P. infestans inoculations, SlPLC2-GFP localizes to the membrane surrounding infection vesicles, where we also detected phosphoinositides PI4P and PI(4,5)P₂. Overexpression of SlPLC2 in Nicotiana benthamiana enhanced susceptibility to P. infestans , reinforcing its positive role in colonization. Together, these findings identify SlPLC2 as a susceptibility factor associated with enhanced P. infestans colonization. Statement This study identifies SlPLC2 as a key susceptibility factor that supports Phytophthora infestans infection by coordinating vesicle formation and early immune responses in tomato.

Extrachromosomal DNA (ecDNA) is a major driver of oncogene amplification, intratumoral heterogeneity and therapy resistance across multiple cancer types, yet there are currently no effective strategies to selectively eliminate it. Here, we show that the type I-E CRISPR system can specifically target and degrade ecDNA in human cancer cell lines (COLO320DM and GBM39). By designing guide RNAs targeting ecDNA-specific breakpoints absent from chromosomal DNA, we achieved efficient depletion of MYC- and EGFR-containing ecDNAs in COLO320DM and GBM39 cells. Loss of ecDNA was accompanied by diminished oncogene signaling, disrupted ecDNA architecture, and impaired tumor cell proliferation, without detectable chromosomal off-target activity. These findings establish a proof-of-concept framework for directly targeting oncogenic ecDNAs and highlight type I-E CRISPR as a promising platform for therapeutic development in ecDNA-driven cancers.

ABSTRACT Cystic fibrosis (CF) is a devastating genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ) gene. As morbidity and mortality from CF results from a lack of mucus clearance that leads to chronic bacterial infections and progressive loss of lung function, site-specific insertion of a CFTR cDNA into the endogenous CFTR locus in airway basal stem cells (ABSCs) could prove curative for all disease-causing mutations. This study describes the development of nonviral genome editing reagents, designed to be packaged into nonviral delivery systems. An sgRNA targeting the 5’ untranslated region of CFTR was characterized as directing high on-target cutting and displaying a safe off-target profile. Airway cell lines electroporated with chemically-modified (1-Aminohexane - AmC6), linear double-stranded DNA (ldsDNA) constructs were utilized as an Homology Directed Repair (HDR) donor, initially optimized with an mCitrine reporter. Expectedly, when the 780bp mCitrine cDNA was replaced with the 4.4kb CFTR cDNA, integration efficiency dropped significantly. However, 1-2% integration of codon optimized donors was sufficient to restore CFTR expression in the bulk edited population of human bronchial epithelial cell line, 16HBE14o- (16HBE), to levels reaching 50% of wildtype expression as measured by Western Blot. Electrophysiological validation of CFTR ion channel function measured via Ussing Chamber Assay revealed that these bulk edited populations exhibit greater than 40% restoration of the chloride ion currents of the measured wildtype controls. These results demonstrate that low levels of CFTR integration can be made therapeutically relevant by optimizing the designs of gene editing reagents. Importantly, this work utilizes nonviral editing reagents, an essential step towards in vivo gene therapy for CF.

In the ciliate Tetrahymena , telomeres of the germline micronucleus (MIC) are removed and replaced by de novo telomere addition during somatic macronuclear (MAC) development. In this study, we investigated the kinetics and mechanism of the MIC telomere elimination. Comparison of the MIC and MAC genome sequences indicated that the MIC telomeres are excised from chromosomes as part of larger MIC-limited sequences (MLSs) through chromosomal breakage. We confirmed this using an optimized oligo-FISH protocol and found that their elimination occurs in parallel with other programmed DNA elimination processes. CRISPR-Cas9 disruption of a MLS-associated Chromosome Breakage Sequence (CBS) showed that elimination of the MLS was not blocked but instead led to loss of adjacent MAC-destined sequence (MDS), suggesting abnormal co-elimination. In biparental crosses of the CBS mutant, however, both MLS and MDS were retained, DNA elimination was broadly disrupted, and no viable progeny were produced. These findings indicate that chromosome breakage at MLS-associated CBSs is essential for the proper separation of MLSs and MDSs, ensuring correct DNA elimination and successful sexual progeny development. We propose that the MIC telomere elimination is subsumed within the broader process of programmed DNA elimination.

The epithelial sodium channel (ENaC) is essential for sodium reabsorption and potassium homeostasis in the distal nephron, where its activity is controlled by mineralocorticoid signaling and downstream proteolytic processing of channel subunits. While cleavage of the γ-ENaC subunit has been implicated in aldosterone-mediated sodium transport, the identity of mineralocorticoid receptor (MR)-regulated proteases responsible for this process remains uncertain. Here, we investigated the role of kallikrein-1 (encoded by Klk1 ), a serine protease expressed in the connecting tubule and cortical collecting duct (CNT/CCD), as a mediator of ENaC activation. Using CRISPR/Cas9, we generated a conditional Klk1 -floxed allele and established mice with CNT/CCD-specific deletion of Klk1 by crossing with Calb1 -Cre (CNT- Klk1 -/- ). On a low sodium, high potassium diet, CNT- Klk1 -/- mice exhibited ∼85% less renal kallikrein-1 expression, yet maintained normal serum electrolytes, urinary potassium excretion, and aldosterone responses. Western blot analysis revealed significantly less cleavage of γ-ENaC and α-ENaC in CNT- Klk1 -/- kidneys, accompanied by more total NCC abundance. Despite impaired ENaC proteolysis, amiloride-sensitive sodium excretion was preserved, indicating intact ENaC function. These findings identify renal kallikrein-1 as a protease that contributes to ENaC subunit processing in vivo . However, the absence of overt sodium or potassium handling defects in CNT- Klk1 -/- mice suggests that kallikrein-1 deficiency is not sufficient to disrupt overall ENaC function, likely due to compensatory mechanisms from redundant proteolytic or non-proteolytic pathways. Together, our results refine the role of kallikrein-1 as a modulator, rather than a sole determinant, of ENaC activation and highlight the complexity of aldosterone-dependent sodium transport in the distal nephron. New & Noteworthy Using a novel connecting tubule / cortical collecting duct specific kallikrein-1 knockout model, we show that γ- and α-ENaC cleavage is impaired by loss of renal kallikrein-1 without major disturbances in sodium or potassium handling. These findings highlight redundancy among ENaC regulatory pathways and suggest that proteolytic cleavage, while biochemically evident, may not be an accurate marker of ENaC-mediated sodium transport under physiological stress.

ABSTRACT Cancer cell evasion of therapy is a highly adaptive process that undermines the efficacy of many treatment strategies. A significant milestone in the study of these mechanisms has been the advent of pooled CRISPR knockout screens, which enable high-throughput, genome-wide interrogations of tumor dependencies and synthetic lethal interactions, advancing our understanding of how cancer cells adapt to and evade therapies. However, the utility of this approach diminishes when applied to dynamic biological contexts, where processes are transient and sensitivity to routine cell culture manipulations that introduce noise and limit meaningful discoveries. To overcome these limitations, we present RESTRICT-seq, a next-generation pooled screening methodology that restricts Cas9 nuclear activation in controlled, repeated cycles. By confining Cas9 catalytic activity to strict temporal windows, RESTRICT-seq mitigates undesired fitness penalties that routinely accumulate throughout pooled screens. When benchmarked against conventional pooled screens and standard inducible protocols, RESTRICT-seq revealed significantly fewer divergent cell clones and increased signal-to-noise ratio, overcoming a key limitation of traditional methods. Leveraging RESTRICT-seq, we conducted a comprehensive functional survey of the druggable mammalian epigenome, uncovering several elusive epigenetic drivers of treatment resistance in cutaneous squamous cell carcinoma (cSCC). This revealed PAK1 as a previously unrecognized mediator of cSCC resistance in human and mouse SCC, offering new insights into a prognostic marker and therapeutic target of high clinical significance. Our findings establish RESTRICT-seq as a powerful tool for extending the applicability of pooled CRISPR screens to dynamic and previously intractable biological contexts.

ABSTRACT Inositol hexakisphosphate kinases (IP6Ks) catalyse the synthesis of the inositol pyrophosphate 5-InsP 7 , and regulate diverse physiological processes. Mice lacking IP6K1 display reduced body weight despite normal food intake, a phenotype that is more apparent in juvenile mice during their rapid growth phase. Additionally, Ip6k1 -/- mice exhibit decreased serum albumin, elevated faecal protein, and reduced skeletal muscle mass compared to Ip6k1 +/+ mice, suggestive of a deficiency in protein digestion in the absence of IP6K1. We found that IP6K1 is expressed throughout the mouse gastrointestinal tract, and is especially enriched in the cytoplasm of chief cells in the stomach, which are responsible for the storage and secretion of digestive enzymes. Pepsinogen C (PGC) containing granules were sparse, and gastric lipase F (LIPF) granules were completely absent in the gastric glands of Ip6k1 -/- mice, despite normal expression levels of these enzymes, implicating IP6K1 in digestive enzyme granule biogenesis. Consequently, the level of the active protease pepsin C was decreased in the gastric lumen of Ip6k1 -/- mice compared with their wild type counterparts. CRISPR/Cas9-mediated deletion of IP6K1 in the gastric adenocarcinoma cell line AGS was able to recapitulate the phenotype of reduced PGC granule intensity seen in gastric chief cells of Ip6k1 -/- mice. PGC granule formation was restored in IP6K1 -/- AGS cells by the reintroduction of catalytically active or inactive IP6K1, indicating that IP6K1 supports the formation of secretory granules independent of its ability to synthesise 5-InsP 7 . The proteoglycan SDC4, identified as an interactor of IP6K1, was seen to co-localise and co-migrate with PGC granules in IP6K1 +/+ but not in IP6K1 -/- AGS cells. Our findings identify IP6K1 as a novel regulator of secretory granule biogenesis in gastric chief cells, to influence protein digestion in the mammalian stomach.

Personalized cancer therapies focus mostly on targeting driver alterations, such as oncogenic point mutations or oncogenic driver events within large somatic copy number alterations. However, these alterations are often not actionable or only present in a small subset of patients. We hypothesized that passenger events, specifically in amplified regions, could be therapeutically exploited by providing actionable molecules on the cell surface, serving as Trojan horses for specific therapy delivery. Applying a multiomics in silico approach, we identified MPZL1 ( Myelin protein zero-like 1 ), a glycosylated cell surface receptor located on chromosome 1q, as a promising candidate, which is amplified in up to 75% of cases in several solid cancers. Notably, immunohistochemistry of a wide range of human cancer tissues (n=2244 samples) as well as normal tissues revealed strong membranous MPZL1 expression in a majority of solid tumors (e.g. 48% of hepatocellular carcinomas or 89% of triple-negative breast cancers), whereas healthy tissues were mostly negative or just faintly positive for MPZL1. Next, we generated a highly specific monoclonal antibody directed to the extracellular domain of human MPZL1 protein and utilized this antibody to produce MPZL1 CAR-T cells. MPZL1 CAR-T cells showed high specificity as well as high sensitivity in targeting a multitude of human cancer cell lines (e.g. liver, breast, and lung cancer) with high MPZL1 expression in vitro. Finally, we demonstrate strong therapeutic efficiency of MPZL1 CAR-T cells not only in different human xenograft tumors in vivo but also in a unique autochthonous liver cancer mouse model. Our work provides a framework to target passenger events within large chromosomal amplifications, reveals MPZL1 as a new trojan horse entry point for therapies of 1q-amplified cancers, and as such opens a new avenue for innovative approaches in anti-cancer drug development.

Chickens ( Gallus gallus ) are uniquely suited for germline studies because their primordial germ cells (PGCs) can be propagated long-term in vitro and used for germline transmission. To develop a loss-of-function screening platform in chicken PGCs, we compared three perturbation methods: CRISPR/Cas9 knockout, CRISPR interference (CRISPRi), and shRNA-mediated knockdown. We found that CRISPR/Cas9 editing causes severe toxicity in PGCs, with DNA damage hypersensitivity over 20-fold greater than in somatic cells, and with distinct DNA damage checkpoint responses between male (ZZ) and female (ZW) lines. CRISPRi using dCas9-KRAB was ineffective in chicken—likely because of species-specific epigenetic constraints—whereas shRNA knockdown produced robust, nontoxic gene silencing. These results identify DNA damage hypersensitivity as a major barrier to nuclease-based editing in the germline and establish RNAi as a feasible platform for genome-wide functional screening in chicken PGCs.

SUMMARY Neurological diseases are frequently characterised by dysregulation of the microtubule cytoskeleton, which is critical for neuronal integrity and functioning. However, the precise mechanisms by which microtubule dynamics are regulated during the development of the nervous system remain poorly understood. Here, we use global phosphoproteomic screening to identify cytoskeletal substrates of Ser-Arg Protein Kinase (SRPK), which is implicated in several neurodevelopmental and neurodegenerative conditions. We show that SRPK directly phosphorylates Microtubule Associated Protein (MAP)1S at multiple sites in a C-terminal region involved in proteolytic maturation and microtubule binding. SRPK-dependent MAP1S phosphorylation modulates the affinity of the MAP1S microtubule binding domain for microtubules and MAP1S proteolytic processing by the Calpain (CAPN)10 protease. Finally, we show that MAP1S proteolytic processing occurs progressively during neurodevelopment via a specific CAPN10 expression switch, corresponding with MAP1S acquisition of microtubule binding activity. Our results demonstrate a role for SRPK in coordinating processing and functionalisation of a key microtubule regulatory protein during neurodevelopment and provide insight into mechanisms by which the microtubule cytoskeleton may be dysregulated in neurological diseases.

Human cytomegalovirus (HCMV) is a prevalent pathogen of the herpesvirus family, infecting most of the human population worldwide. Like all herpesviruses, HCMV can establish a latent infection that persists throughout the lifetime of the host. The HCMV immediate early (IE) proteins, IE1 and IE2, are viewed as master regulators of HCMV infection and are commonly assumed to play pivotal roles in regulating the balance between latent and lytic infection, as their repression is a hallmark of latency. However, it is still unclear whether their expression can indeed determine the establishment of productive infection and what functions, either related to viral gene expression or to cellular pathways, are involved in this activity. Using THP1 monocytes, ectopically expressing the HCMV receptor, PDGFRα to boost viral entry, we show that overexpression of either IE1 or IE2 significantly enhances productive infection, illustrating their critical role in determining infection outcome. Mechanistically, we show IE2 drives expression of the viral early genes at early stages of infection, whereas IE1 acts more broadly to enhance global viral gene expression. We further show that from the many functions of IE1, its ability to promote lytic infection is mainly linked to the disruption of PML nuclear bodies. Importantly, induction of either IE1 or IE2 expression in latently infected cells enhances viral reactivation, with IE1-mediated PML representing a central mechanism. Taken together, our findings elucidate the distinct and complementary roles of IE1 and IE2 in overcoming barriers to productive infection and reactivation.

ABSTRACT The human heart, originating from the splanchnic mesoderm, is the first functional organ to develop, co-evolving with the foregut endoderm through reciprocal signaling. Previously, cardioid models offered new insights on cardiovascular cell lineages and tissue morphogenesis during heart development, while mesoderm-endoderm crosstalk remain incompletely understood. Here, we integrated micropatterned cardioids, CRISPR-engineered reporter hiPSCs, deep-tissue imaging, and single-cell RNA sequencing (scRNA-seq) to explore synergistic mesoderm-endoderm co-development. scRNA-seq with PHATE trajectory mapping reconstructed lineage bifurcations of mesoderm-heart and endoderm-foregut lineages, identifying key cell types in cardiac and hepatic development. Ligand-receptor interaction analysis highlighted mesodermal cells enriched in non-canonical WNT, NRG, and TGF-β signaling, while endodermal cells exhibited VEGF and Hedgehog activity. We found that micropattern sizes influenced cellular composition, cardioid cavitation, contractile functions, and mesoderm-endoderm signaling crosstalk. The cardioids generated from 600 µm diameter circle patterns showed larger cavity formation resembling early heart chamber formation. Our findings establish micropatterned cardioids as a model for mesoderm-endoderm co-development, enhancing our understanding of heart-foregut synergy during early embryogenesis.

The CRISPR-Cas system provides adaptive immunity in many bacteria and archaea by storing short fragments of viral DNA, known as spacers, in dedicated genomic arrays. A longstanding question in CRISPR-virus coevolution is the optimal number of spacers for each bacterium to maintain proper phage coverage. In this study, we investigate the optimal CRISPR memory size by combining steady-state immune models with dynamical antigenic traveling wave theory to obtain both analytic and numerical results of coevolutionary dynamics. We focus on two experimentally supported phenomena that shape immune dynamics: primed acquisition, where partial spacer-protospacer matches boost acquisition rates, and memory size fluctuations, where a short-term increase in memory size drives population dynamics. We find that under primed acquisition, longer optimal arrays benefit from maintaining multiple, partially matching spacers. In contrast, dynamic memory fluctuations favor shorter arrays by amplifying the fitness advantage of acquiring a few highly effective new spacers. Together, our results highlight that memory optimality is not fixed, but instead shaped by the interaction of acquisition dynamics and population-level immune pressures.

Natural killer (NK) cell-based immunotherapies represent a promising avenue for cancer treatment due to their ability to eliminate cancer cells independently of antigen presentation and potential for “off-the-shelf” use. However, the molecular determinants governing tumor cell susceptibility to NK cell-mediated cytotoxicity remain incompletely understood. Here we employed CRISPR activation (CRISPRa) screening to systematically identify cancer cell surface regulators of NK cell killing across multiple cancer types. Using a comprehensive surfaceome-focused library, we screened human and murine cancer cell lines co-cultured with NK cells, identifying both known and novel ligands that modulate NK cell cytotoxicity. Our screens revealed established factors including CD43 (encoded by SPN ), while uncovering previously uncharacterized regulators such as CD44, PDPN, and Siglec-1/CD169. Validation through complementary cDNA overexpression and genetic knockout approaches confirmed that disruption of CD43, CD44, PDPN, and Siglec-1 significantly altered cancer cell susceptibility to NK killing both in vitro and in humanized mouse models. Analysis of clinical datasets show that expression of identified factors correlates with patient survival outcomes in an NK-context dependent manner supporting their therapeutic relevance. Most notably, our mechanistic studies demonstrate that CD43-mediated NK cell resistance operates independently of its previously proposed interaction with Siglec-7 on NK cells. Furthermore, we find that targeting CD43 on either NK cells or engineered T cells substantially enhances their cytotoxic activity against leukemia cell lines. These results establish gain-of-function screening as a powerful approach for discovering immunoregulatory surface proteins and identify multiple promising targets for enhancing NK cell-based cancer immunotherapies.

Abstract Biologics produced in Escherichia coli BL21(DE3) require rigorous removal of lipopolysaccharide (LPS), also termed endotoxin, as its presence can elicit severe immune responses and life threatening complications in humans. Escherichia coli Nissle 1917 (EcN) is a clinically approved probiotic with unique biosafety characteristics due to its LPS-deficient phenotype, and has only 0.86% of the LPS activity observed in BL21(DE3), so its markedly lower immunostimulatory potential makes it an attractive chassis for low-LPS protein production. Using GFP as a model protein for comparison, its yield in EcN is only ~ 30% of that observed in BL21(DE3) owing to lower cell density (OD₆₀₀) and per-cell protein yield. Transcriptomic profiling revealed that heterologous protein induction in EcN suppresses key biosynthetic and energy-generating pathways—including ribosome biogenesis, translation, and the tricarboxylic acid (TCA) cycle—thereby constraining heterologous protein synthesis. To boost intracellular protein synthesis in EcN, we first inserted the T7 RNA polymerase gene into the chromosome, creating EcN::T7. We then used CRISPR/Cas9-mediated genome editing technology to delete ompT, iclR, and arcA, yielding the high-yield mutant EcN::T7Δ ompT Δ iclR Δ arcA . This engineered strain produced 3.2-fold more reporter protein than its parental strain EcN::T7, reaching ~ 70% of the yield observed in BL21(DE3). When applied to interferon α-2b (IFNα-2b) production, mutant EcN::T7Δ ompT Δ iclR Δ arcA achieved 89.3% of BL21(DE3)’s titer while exhibiting post-purification LPS levels comparable to BL21(DE3). Notably, the the IFNα-2b retained full biological activity. By eliminating the need of extensive LPS removal procedures, this strategy positions EcN as a cost-effective and clinically compliant platform for biomanufacturing.

A bstract Whole genome doubling (WGD) is a frequent event in tumourigenesis that promotes chromosomal instability and tumour evolution. WGD is sensed indirectly due to the presence of extra centrosomes, which activate the PIDDosome to induce a p53-dependent G1-arrest. Here we uncouple WGD from centrosome amplification and show that p53 still arrests tetraploid cells, but via the mitotic stopwatch; a p53/53BP1/USP28-dependent pathway that causes G1-arrest following an extended mitotic delay. Mitotic timing is unaffected by WGD, but the threshold mitotic delay needed to invoke a G1-arrest is reduced. This sensitivity to the stopwatch mechanism is not associated with altered levels of mitotic stopwatch components, but instead, is associated with enhanced p21 concentrations prior to mitosis. Similar effects are observed in diploid cells treated with CDK4/6 inhibitors to double their size, implicating G1 delays and cell size as key determinants of stopwatch sensitivity. This ability of the mitotic stopwatch to arrest proliferation after increases to genome or cell size has important implications for the progression and treatment of cancer. It also demonstrates that the stopwatch pathway can sense more than just mitotic delays.

Neuropilin-1 (NRP-1) is a versatile transmembrane protein expressed in numerous cell types and tissues, both in health and in disease. In particular, it is expressed by activated T cells, where it has been shown to play an inhibitory role, dampening their response to tumor cells. Chimeric antigen receptor (CAR) T cells have had considerable success in treating cancers such as B cell leukemias, but face several obstacles in the treatment of solid tumors. We hypothesized that NRP-1 may contribute to dampening CAR T cell responses to cancer. While NRP-1 blockade either through neutralizing antibodies or through CRISPR/Cas9-mediated deletion did not improve CAR T cell cytotoxic activity in an in vitro solid tumor model, NRP-1 was found to have a protective effect against CAR T cells when expressed by the tumor cells, in line with previous literature implicating NRP-1 as a pro-tumor factor involved in their growth and invasiveness. We found that NRP-1 was transferred from target cells to CAR T cells via trogocytosis, i.e. the “nibbling” and subsequent display of membrane proteins from one cell by another. CD19, the target antigen in this model, was also transferred to CAR T cells, raising interesting questions about trogocytosis as a marker for effective tumor cell killing through direct interaction. On the contrary, trogocytosis may impede effective CAR T function by promoting fratricide due to their display of target antigen. While no conclusive mechanisms for NRP-1 activity were found, several avenues of research have been opened up to further our understanding of the multifaceted roles of NRP-1 in CAR T cell activity and interactions with their targets.

Genetic variation within species shapes phenotypes, but identifying the specific genes and variants that cause phenotypic differences is costly and challenging. Here, we introduce CRI-SPA-Map, a genetic mapping strategy combining CRISPR-Cas9 genome engineering, selective ploidy ablation (SPA), and high-throughput phenotyping for precise genetic mapping with or without genotyping in the yeast Saccharomyces cerevisiae . In CRI-SPA-Map, a donor strain carrying SPA machinery is mated to a genetically different recipient strain harboring a genome-integrated selectable cassette. In the resulting diploid, CRISPR-Cas9 cuts the cassette for replacement with DNA from the homologous donor chromosome. Donor chromosomes are then removed using SPA to yield haploid recombinant strains. To establish CRI-SPA-Map, we mated a W303 SPA strain to 92 strains from the BY4742 yeast knockout collection that carry gene deletion cassettes on the left arm of chromosome XIV and created 1,451 recombinant isolates. Whole-genome sequencing verified that deletion cassette replacement introduced short donor DNA tracts of variable length, resulting in a finely recombined mapping population. Using only the known location of the gene deletions, which marks where donor DNA is introduced, we identified a 6.5 kb-region shaping yeast growth. Further dissection of this region pinpointed two causal variants in two genes, MKT1 and SAL1 . Engineering these variants alone and in combination revealed gene-by-environment interactions at both genes, as well as epistatic interactions between them that were in turn dependent on the environment. CRI-SPA-Map is a cost-effective strategy for creating high-resolution recombinant panels of yeast strains for identifying the genetic basis of phenotypic variation.

To grow and divide cells must tightly coordinate anabolic programs with the availability of nutrients and growth factors. This balance is especially critical during postnatal development, when biosynthetic and energetic demands are high, and nutrient supply and neonates have to adapt to periods of fasting. These conditions place acute stress on the proteostasis network, making autophagy essential for nutrient recycling. We found that the chaperone aryl hydrocarbon receptor-interacting protein (AIP) supports both arms of this metabolic balance: promoting anabolic PI3K-AKT signaling for mTORC1 activation and enabling catabolic processes such as proteasomal degradation and autophagy. Loss of AIP causes a severe neonatal metabolic disorder, where affected infants fail to thrive postnatally. Our findings establish AIP as a central regulator of neonatal metabolic adaptation and cellular homeostasis. One Sentence Summary AIP integrates nutrient sensing and protein recycling to sustain neonatal survival.