ABSTRACT Carbon and zinc (Zn) metabolism are intrinsically connected in phototrophs, as crucial components involved in CO 2 assimilation, like carbonic anhydrases, are highly abundant Zn proteins. Utilizing these and other proteins, the eukaryotic green algae Chlamydomonas reinhardtii can maintain phototrophic growth in low CO 2 environments by inducing a carbon concentrating mechanism (CCM). In this work we show that Chlamydomonas dynamically increases its Zn content to accommodate the higher intracellular Zn demand in low CO 2 environments. This increase requires the presence of Cia5, a major regulator of the CCM in Chlamydomonas. How Cia5 regulates expression of thousands of low CO 2 -inducible genes remains enigmatic, its transcript and protein abundance is unchanged in different CO 2 environments, even in the presence of an additional reduced carbon source, acetate. We show here that the Cia5 protein is not present in Zn-limitation, despite CIA5 transcription being unchanged. We used a CRISPR knock-in approach to express Cia5-HA from its endogenous locus and used two independent Cia5-HA expressing strains for affinity purification and identified a protein belonging to a conserved family of metal binding GTPases, ZNG3, as a constitutive interaction partner. Like Cia5, ZNG3 is constitutively expressed, co-expressed with Cia5 along the diurnal cycle and is Cia5-dependently induced in low CO 2 environments. Surprisingly, zng3 mutants do not phenocopy cia5 mutants and grow well in low CO 2 conditions. Instead, zng3 mutants are unable to grow like wildtype if excess carbon is available in the form of high CO 2 or acetate. Transcriptomics of wildtype and zng3 mutants grown with different carbon sources revealed that transcriptional induction of the majority of genes involved in the CCM is maintained in low CO 2 grown zng3 mutants, while the degree of induction in a subset of LCI genes is reduced ( HLA3 , CAH4 and CAH5 ). Genes encoding proteins involved in plastid quality control were induced in zng3 mutants grown on acetate and high CO 2 , as well as other, related metallochaperones. We hypothesize that Zn trafficking towards the plastid is mis regulated in zng3 mutants resulting in protein mis-metalation and unfolding. Taken together, we propose that ZNG3 and Cia5 coordinate Zn and CO 2 metabolism, affecting intracellular Zn trafficking and modulate the CO 2 response.

ABSTRACT Typically diagnosed late, when systemic metastasis has already occurred, pancreatic ductal adenocarcinoma (PDAC) has one of the worst 5-year survival rates of any cancer type. For many patients with advanced disease, current chemotherapy regimens offer only modest benefit despite significant toxicity and surgical resection, the only treatment option with curative potential, is not possible. Therefore, while new treatments are much needed, diagnosing patients at an ‘earlier’ disease stage when surgery remains possible and the window of opportunity for treatment response is longer will be critical to improving patient outcomes. In this regard, the identification of biomarkers from biospecimens that can be easily sampled from patients remains the focus of considerable research, however success has not been forthcoming. Using a suite of novel genetically defined murine isogenic models of early PDAC, engineered using CRISPR-Cas9 gene editing, we sought to address whether loss-of-function mutations in common driver genes, and thus the genetic heterogeneity inherent to the disease, may represent an important confounding factor in the identification of a one-size-fits-all biomarker suitable for early detection. Focussing on the multi-omics analysis of blood, we show that both loss of Cdkn2a and / or Smad4 on the background of a Kras G12D Trp53 -/ - genotype has profound effects on the profile of differentially expressed RNA species including protein coding RNAs, lncRNAs, snoRNAs, scRNAs, snRNAs and miRNAs, and on plasma protein expression, when compared to both healthy controls and chemically induced pancreatitis. In addition, we find that loss of Smad4 , a genomic event that occurs following progression from PanIN to PDAC, substantially limits the availability of blood biomarkers. These findings identify the need to move towards genotype-specific biomarker signatures and uncover a potential role for Smad4 loss in limiting opportunities for the early detection of pancreatic cancer.

Since 2021, subclade 2.3.4.4b A(H5N1) high pathogenicity avian influenza (HPAI) viruses have undergone changes in ecology and epidemiology, causing a panzootic of unprecedented scale in wild and domestic birds with spill-over infections and perceptible transmission in a range of mammalian species, raising concern over zoonotic potential. HPAI viruses readily exchange gene segments with low pathogenicity avian influenza viruses via reassortment, a mechanism that facilitates pronounced phenotypic change. Observations suggest changes in the seasonality and host range of panzootic viruses, however, data on the role of reassortment in determining such features are limited. Using phylodynamic approaches, we describe the emergence of the panzootic lineage and using a novel global genotype classification system we describe the subsequent emergence and global structuring of genotypes generated by reassortment. Focusing on evolutionary dynamics in Europe, we show reassortment has produced high fitness genotypes with enhanced capacity for transmission and further we show such advantages can be host-dependent, contrasting successful generalist genotypes with a specialist lineage (EA-2022-BB) adapted to birds of the order Charadriiformes. Experimental investigation of NS1-mediated shutoff indicates this Charadriiformes-specialist does not inhibit host cellular gene expression and hamper the defences of more typical hosts such as water- and land-fowl. We attribute this primarily to variation at position 127 of the NS1 protein. Our results emphasise that reassortment has driven phenotypic change, affected viral fitness, and caused diversification of host specificity and seasonality. Such factors should be considered in studies that seek to identify drivers of HPAI spread and map spillover risk. Additionally, relaxation of host specialisation, ecological diversification, and potential endemicity in atypical host populations present new reassortment opportunities that could result in further novel phenotypes.

Abstract Pooled CRISPR-Cas9 screens have emerged as powerful functional genomic tools. However, they typically rely on aggregate-level or transcriptomic outputs, which limits their ability to capture spatially resolved phenotypes. We developed spatial phenotype analysis of CRISPR-Cas9 screens (spaCR), an open-source software package that integrates deep-learning single-cell image classification with barcode-based, well-level genotype mapping to link pooled genetic perturbations to spatial phenotypes. Unlike optical pooled screening or in situ genotyping methods, spaCR requires only standard imaging and sequencing platforms, eliminating the need for specialized equipment. To guide experimental design, we developed spaCRPower, a statistical simulation tool that models screen parameters and estimates power for detecting genotype–phenotype associations. As a proof-of-concept, we applied spaCR to a pooled Toxoplasma mutant library to identify parasite effectors involved in hijacking the host ESCRT machinery. We identified both an established and a previously uncharacterized genetic determinant, EAF1, highlighting spaCR as a powerful tool for mapping genotype–phenotype relationships.

Abstract Autoimmune diseases represent a class of complex, multifactorial disorders characterized by the immune system’s aberrant recognition of self-antigens as foreign, leading to chronic inflammation, tissue damage, and progressive organ dysfunction. Despite advances in immunosuppressive pharmacology, current therapies remain largely non-specific, systemically cytotoxic, and incapable of achieving long-term immune tolerance. In response to this therapeutic gap, we present SynAuto-X—a synthetic biology platform integrating a programmable CRISPR-dCas9-based gene circuit designed for the conditional silencing of pro-inflammatory genes that drive autoimmune pathology. SynAuto-X employs a modular genetic architecture based on catalytically inactive Cas9 (dCas9) fused to the KRAB (Krüppel-associated box) repressor domain. This fusion enables precise transcriptional repression of key cytokine and co-stimulatory genes (e.g., IL17A, IFNG, TNFA, CD80) through CRISPR interference (CRISPRi), directed by customizable single-guide RNAs (sgRNAs). Gene silencing is driven by inflammationresponsive promoters, such as NF-B or STAT1 elements, allowing context-specific activation of the repression cascade only in immune cells undergoing aberrant activation. The architecture supports single-input singleoutput (SISO) and combinatorial logic designs, enabling multigene targeting, threshold-dependent activation, and Boolean gating of inflammatory response elements. A key innovation within SynAuto-X is the integration of a drug-inducible kill switch for enhanced biosafety. Utilizing a chemically controlled inducible Caspase-9 (iCasp9) domain responsive to the synthetic small molecule AP1903, the system enables externally triggered apoptosis of transduced cells upon detection of adverse immune modulation or unintended off-target effects. The dynamics of this safety module are modeled through coupled differential equations, representing CID-mediated dimerization and apoptotic cascade activation, with tunable parameters for dose-responsive control. Additionally, the full circuit is encoded within a lentiviral vector framework, optimized via Benchling and CHOPCHOP for minimal off-target sgRNA activity, promoter leakiness, and vector immunogenicity. SynAuto-X represents a programmable, safe, and cell-specific alternative to broad-spectrum immunosuppression. By combining precision CRISPR interference, logic-based gene circuit design, and fail-safe therapeutic control, SynAuto-X advances the frontiers of autoimmune treatment toward intelligent, synthetic immune regulation. This work lays the foundation for a new class of gene circuit-based therapeutics capable of rewiring immune responses with the specificity of molecular computation and the safety of on-demand self-destruction.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates apoptosis, autophagy, inflammation, and intracellular pathogen survival. While S1P signaling has been implicated in Leishmania donovani infection, the specific roles of its biosynthetic enzymes Sphingosine kinases SphK1 and SphK2 in host macrophage remains poorly defined. Here, we delineate the role of SphK1 in modulating host-pathogen interactions using CRISPR/Cas9-mediated knockout and pharmacological inhibition in THP-1 macrophages. We generated and validated CRISPR constructs targeting SphK1 (promoter, SBS, exon 6) and SphK2 (exon 3). SphK1 Knockout was confirmed at transcript and protein levels, accompanied by a marked reduction in SphK1 enzymatic activity and S1P levels. Functionally, SphK1 knockout macrophages exhibited decreased intracellular L. donovani burden, elevated TNF-α, and reduced IL-10, and increased autophagic and apoptotic markers, suggesting a pro-inflammatory, cell-death-prone state. Pharmacological inhibition using the selective SphK1 inhibitor, PF-543 recapitulated these findings, showing reduced phosphorylated SphK1, enhanced p38MAPK activation, and augmented autophagy and apoptosis. Conversely, the SphK2 inhibitor ABC294640 had minimal effect, reinforcing the predominant role of SphK1. Together, our study identifies SphK1 as a critical host factor that facilitates L. donovani survival by modulating lysosomal stress, immune evasion, and cell fate pathways. Targeting SphK1-S1P signalling may offer a novel therapeutic approach for visceral leishmaniasis.

Mounting evidence implicates microglia in neurodegeneration, but linking disease-associated genetic variants to target genes and cellular phenotypes is hindered by the inaccessibility of these cells. We differentiated 261 human iPSC lines into microglia-like cells (iMGL) in pools with phenotypic (differentiation, phagocytosis and migration) and single-cell transcriptomic readouts. Burden analysis of deleterious variants detected 36 genes influencing microglial phenotypes. Expression quantitative trait locus (eQTL) analysis found 7,121 eGenes, and 79 colocalizations across four neurodegenerative disease GWAS, half of which had limited prior evidence of causality. Integration of eQTL and phenotypic associations highlighted the role of disease-relevant variants including LRRK2 and TREM2 acting via microglial phagocytosis. A coupled CRISPR screen identified a role of TREM2 in phagocytosis and highlighted the importance of cellular state in directionality of phenotype. By contextualizing variant effects within disease-relevant microglial states, we provide a comprehensive framework for interpreting the function of risk loci in neurodegenerative disorders.

ABSTRACT The emergence of SARS-CoV-2 has posed significant threats to global health, particularly for the older population. Similarly, common human coronaviruses, such as HCoV-229E, which typically cause mild cold-like symptoms, have also been linked to severe diseases, underscoring the need to understand virus-host interactions and identify host factors contributing to viral pathogenesis and disease progression. In this study, we performed a genome-wide CRISPR knockout screen using HCoV-229E and identified Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) as a potent restriction factor. Mechanistically, UHRF1 suppressed HCoV-229E infection by downregulating the expression of its cell entry receptor, aminopeptidase N (APN), through promoter hypermethylation. Focused CRISPR activation screens of UHRF1-downregulated genes confirmed the critical role of APN in HCoV-229E infection and identified additional genes (e.g., SIGLEC1, PLAC8, and heparan sulfate biosynthesis genes) contributing to the restrictive functions of UHRF1. Transcriptomic and single-cell RNA sequencing analysis revealed that UHRF1 expression decreases with age, negatively correlating with increased APN expression. This age-related decline in UHRF1 was further validated in primary alveolar macrophages isolated from elderly individuals, which exhibited heightened susceptibility to HCoV-229E infection compared to those from younger individuals. Our findings highlight UHRF1 as a key age-related host defense factor against coronavirus infection and provide novel insights into the epigenetic regulation of viral entry receptors.

Sporozoites of Plasmodium falciparum , the deadliest malaria parasite, are transmitted into the skin by infected mosquitoes and migrate to the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that, eventually, enter the bloodstream and invade erythrocytes, leading to malaria. The parasite journey involves cell traversal, where sporozoites transiently enter and exit host cells beginning in the skin, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish liver-stage infection. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host determinants. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining in HC-04 Cas9+ (clone 2B3) cells. Optimized traversal and invasion assays with HC-04 2B3 cells led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. As proof-of-concept, we performed a small screen involving disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens, confirming utility of this platform. While no new host factors were identified for malaria parasites in this initial study, we have developed a tractable system for genome-wide CRISPR screens to uncover novel hepatocyte biology and host determinants of infection by liver-tropic pathogens.

SOCIETAL STATEMENT EN - Goldenberry ( Physalis peruviana ) produces sweet, nutritionally-rich berries, yet like many minor crops, is cultivated in limited geographical regions and has not been a focus of breeding programs for trait enhancement. Leveraging knowledge of plant architecture-related traits from related species, we used CRISPR/Cas9-mediated gene editing to generate a compact ideotype to advance future breeding efforts and agricultural production. Goldenberry growers will benefit from these compact versions because it optimizes per plot yield, facilitating larger-scale production to meet rising consumer popularity and demand. SP - La uchuva ( Physalis peruviana ) produce frutos dulces y ricos en nutrientes, pero, igual que muchos cultivos minoritarios, se cultiva en zonas geográficas limitadas y no ha sufrido un proceso de mejora. Aprovechando conocimientos sobre rasgos relacionados con la arquitectura vegetal de especies relacionadas, hemos usado edición génica mediante CRISPR/Cas9 para generar un ideotipo compacto para promover futuros esfuerzos en su mejora y en producción agrícola. Los productores de uchuva se podrán beneficiar de estas versiones compactas ya que optimiza el rendimiento por parcela, facilitando así la producción a una mayor escala para cubrir la creciente popularidad y demanda de los consumidores.

Abstract Insulin-like growth factor 1 (IGF-1) is known to promote cancer cell proliferation, but its role in metastasis remains incompletely understood. Autophagy, a key regulator of cancer cell behavior, plays a significant role in colorectal cancer (CRC) progression. Our previous transcriptomic analysis identified autophagy-related genes and insulin-like growth factor 1 receptor (IGF-1R) among the most differentially expressed in advanced versus early-stage CRC. However, the mechanistic contribution of IGF-1R to autophagy-driven CRC metastasis has not been fully elucidated. In this study, we investigated the functional interaction between IGF-1R signaling and autophagy in CRC progression using a panel of CRC cell lines, including HCT116 cells with CRISPR/Cas9-mediated knockout of ATG5 and ATG7. Our results demonstrate that IGF-1 stimulation enhances autophagic flux, whereas IGF-1R knockdown suppressed autophagic activity. Notably, dual inhibition of IGF-1R and autophagy led to a marked reduction in CRC cell migration and invasion. In ATG5-/- and ATG7-/- cells, IGF-1R silencing significantly downregulated mesenchymal markers Vimentin, Slug, and Snail while upregulating the epithelial marker E-cadherin. Additionally, combined inhibition increased size and number of focal adhesion molecules, such as paxillin and zyxin. These findings highlight the synergistic effect of IGF-1R and autophagy inhibition in suppressing EMT and metastatic potential in CRC cells, suggesting that this combinatorial approach may represent a promising therapeutic strategy for metastatic CRC. Further studies are warranted to delineate the underlying molecular mechanisms and evaluate these findings' translational potential in clinical settings.

Abstract Gaucher disease type 1 is a lysosomal storage disorder caused by GBA1 mutations that reduce glucocerebrosidase activity, leading to glycolipid buildup, particularly in macrophages. To develop a curative approach, we established a high-efficiency genome editing platform for human and murine hematopoietic stem-progenitor cells using CRISPR/Cas9, recombinant adeno-associated virus serotype 6. To enhance homology-directed DNA repair while minimizing genotoxicity, we incorporated a new 53BP1 inhibitor, a ubiquitin variant that promotes DNA end resection and significantly increases editing efficiency. This enabled precise insertion of a human GBA1 transgene—driven by a macrophage-specific promoter—into the mouse Rosa26 and human CCR5 safe-harbor loci. To assess efficacy, we established a rapidly progressive Gaucher disease mouse model by inducing hematopoietic-specific Gba1 deletion in a D427V background. Transplantation of edited cells corrected hematologic and visceral abnormalities, normalized lipid storage, and was effective under myeloablative and reduced-intensity busulfan conditioning. Notably, therapeutic benefit was achieved with only ~ 3% edited allele engraftment. These findings offer strong proof-of-concept for ex vivo genome editing as a mutation-agnostic, potentially curative strategy for Gaucher disease and support its clinical advancement.

Autophagy degrades cellular material by sequestering it in autophagosomes, which form de novo from precursors called phagophores. Phagophore assembly and expansion require ATG9A-positive seed compartments, the lipid transfer protein ATG2A, and the class III phosphatidylinositol 3-phosphate kinase complex I (PI3KC3-C1). PI3KC3-C1 synthesizes phosphatidylinositol 3-phosphate (PI3P), a key lipid that drives downstream processes for phagophore expansion, including ATG8 lipidation. We find that ATG9A compartments contain only traces of phosphatidylinositol (PI), likely insufficient for efficient PI3P production or recruitment of PI3P-binding effectors. Nevertheless, ATG2A is recruited to these compartments and mediates lipid transfer, including PI, into them. Remarkably, even without detectable PI3P, ATG9A compartments can support ATG8 lipidation, and ATG8 proteins themselves enhance ATG2A-mediated lipid transfer. In cells, ATG2A is essential for the appearance of PI3P on ATG9A compartments. Together, these findings support a model in which a lipid transfer-driven feedback loop, rather than pre-existing PI content, is required to activate ATG9A compartments for phagophore expansion. Teaser A feedback loop driven by lipid transfer activates ATG9A compartments for autophagosome biogenesis.

The isomerohydrolase RPE65 is a critical element of the visual cycle, the series of enzymatic reactions by which the chromophore of the visual pigments is regenerated following light exposure. In humans, mutations in the rpe65 gene cause a severe form of blindness called Leber’s congenital amaurosis. Studies of RPE65 -/- mice have shown dramatic depletion of 11- cis-retinal in the retina, resulting in a slow retinal degeneration. However, a number of studies suggest that RPE65 may not be necessary for the regeneration of photopigment in all photoreceptor types. Using CRISPR/Cas9 technology, we previously generated RPE65 knockout Xenopus laevis in order to test the involvement of rhodopsin chromophore in the cell death mechanisms associated with rhodopsin mutations and rhodopsin quality control. Here we further characterize the effects of RPE65 knockout in these animals, and show their rod photoreceptors have shortened outer segments that lack detectable rhodopsin photopigment. However, there is no progressive degeneration of rods or cones. Via electroretinography we found greatly reduced but significant responses to light under scotopic and photopic conditions. We also found reduced behavioral sensitivity to light, while light-induced melanophore dispersion was unaffected. RPE65 knockout X. laevis may be a useful system for examining RPE65-independent photosensation mechanisms in vertebrates.

Most microbial taxa on Earth remain uncultivated, limiting our ability to study their physiology, ecology, and roles in environmental processes. Although metagenome-assembled genomes (MAGs) have expanded access to uncultured phylogenetic diversity, the functional basis for culturability remains poorly understood. Here, we analyze the 52,515 MAGs from the Genomes from Earth’s Microbiomes (GEM) catalog to test two hypotheses: 1) genomes from uncultured microbes encode more functionally novel genes than those from cultured taxa, and 2) specific genomic features are systematically associated with culturability across phyla. To assess functional novelty, we aligned predicted proteins to SwissProt and measured sequence dissimilarity to the nearest curated homolog. We find that uncultured MAGs, particularly among Archaea, harbor substantially more divergent proteins. To identify genomic traits predictive of culturability, we combined pathway-level enrichment with LASSO regression and permutation-based feature importance. Cultured MAGs were consistently enriched in Clusters of Orthologous Groups (COG) pathways related to vitamin and cofactor biosynthesis (e.g., thiamine, folate, B12), energy metabolism (e.g., TCA cycle), and CRISPR-Cas systems—functions often depleted in uncultured counterparts. LASSO models identified a subset of these pathways as strong predictors of cultured status even in poorly sampled phyla, suggesting conserved genomic signatures of culturability. In contrast, pathways such as purine biosynthesis and NADH dehydrogenase were associated with uncultured lineages, highlighting potential barriers to cultivation. These results 1) demonstrate the great functional novelty of uncultured microbes, potentially offering unprecedented opportunities for discoveries of novel function, and 2) identify metabolic traits associated with culturability to inform future cultivation strategies. Importance The vast majority of microbes are uncultured, which means they have never been characterized under laboratory conditions. We showed that genomic sequences of uncultured microbes have less similarity to characterized proteins compared to cultured microbes, revealing that there may be fundamental biological reasons why they are not cultured. We also showed that certain metabolic pathways, such as those related to vitamin and cofactor biosynthesis, can predict the ability of microbes to grow under laboratory conditions, and these pathways are abundant in highly cultured phyla, indicating how metabolic pathways can influence cultivation strategies.

Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major , localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9–mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (Sb III ), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in Sb III EC 50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance. Author Summary Leishmaniases are neglected tropical diseases caused by parasites that survive and multiply inside vertebrates’ cells. These parasites rely on hosts’ nutrients like iron and must resist both host defenses and treatment with toxic drugs such as antimony. We studied a protein called Aquaglyceroporin 1 (AQP1) in Leishmania amazonensis , a species that causes skin lesions in South America. Unlike related species, where AQP1 is found on the parasite’s surface, we discovered that in L. amazonensis AQP1 is located in an internal organelle called glycosome. By deleting this protein from the parasite, we found that it plays a crucial role in iron balance, sensitivity to antimony drugs, and the parasite’s ability to cause disease. Unexpectedly, parasites without AQP1 were more resistant to antimony but still accumulated high levels of the drug, suggesting that Leishmania has other ways of taking up antimony. Our findings challenge the assumption that all Leishmania species use the same strategies to survive, and highlight the need to understand species-specific differences when designing treatments or analyzing parasite biology.

SUMMARY Metastasis is an emergent continuum, driven by evolving reciprocal adaptations between continuously disseminating tumor cells (DTCs) and the specialized metastatic niches of distant organs. The interplay between intrinsic and niche-driven mechanisms that enables DTCs to survive and home to distant organs remains incompletely understood. Here, using MetTag, a single-cell barcoding and transcriptome profiling approach with time-stamped batch identifiers (BC.IDs) and functional CRISPR screening, we resolved the clonality, temporal dynamics, and molecular determinates of DTC colonization success across evolving metastatic niches. Deep sequencing of barcodes revealed preferred enrichment of early-disseminated clones across metastatic niches. Single-cell RNA sequencing (scRNA-seq) coupled with RNA velocity analyses in ascites and metastasis-bearing omenta uncovered an emergent and distinct interferon-gamma (IFNγ) centric transcriptional trajectory, enriched among early seeding clones. In vivo CRISPR/Cas9 screening of metastatic niche-specific signatures demonstrated that genes belonging to the IFNγ response are functionally important for peritoneal metastasis. Knockout of IFNγ receptor 1 ( Ifngr1 ) in the first batch of DTCs significantly reduced metastatic burden and extended survival, underscoring the importance of tumor cell intrinsic IFNγ signaling in shaping post-seeding metastatic niche (PSMN) and subsequent metastatic co-evolution. Mechanistically, tumor intrinsic IFNγ response and ascites-derived tumor-associated macrophages (TAMs) protect cancer cells from anoikis-mediated death by promoting pro-survival signaling. Our study defines temporal dynamics of disseminating tumor cells at metastatic niches and reveals a general “first come, first served” pro-metastatic adaptation principle of DTCs. Graphical abstract

ABSTRACT Germline pathogenic BRCA1 variants predispose women to breast and ovarian cancer. Despite accumulation of functional evidence for variants in BRCA1 , over half of reported single-nucleotide variants (SNVs) lack a definitive clinical interpretation. Furthermore, the extent to which variant effects are consistent across cell types remains largely unexplored. Here, we performed saturation genome editing (SGE) of BRCA1 in HAP1 cells to score 4,113 variants not previously assayed. Additionally, we developed a new SGE assay in human mammary epithelial cells (HMECs), allowing effects of variants to be compared across cell lines, drug treatments, and genetic backgrounds. We identify 538 variants impacting function via diverse mechanisms, including impairment of the BRCA1–PALB2 interaction and disruption of splicing, transcription, and translation. Function scores from experiments in HAP1 discriminate known pathogenic and benign variants with near-perfect accuracy. Intriguingly, however, nearly half of variants impacting function in HAP1 were found to be neutral when assayed in HMECs. We show that discordantly scored variants are hypomorphic and confer intermediate cancer risk. These results will be highly valuable for clinical interpretation of BRCA1 variants. Moreover, this work illustrates how revealing context-specific variant effects across cell types can enable more accurate resolution of disease risk.

ABSTRACT While NLRP3 has been extensively studied in myeloid cells, its existence and regulation in epithelial cells, including keratinocytes, are unclear. In fact, whether human keratinocytes express a functional NLRP3 inflammasome at all remains a matter of debate in the inflammasome field. Here, we provide additional evidence that NLRP3 is repressed in human keratinocytes cultured under non-inflammatory conditions but can be sharply induced by interferon-γ (IFNγ)—but not lipopolysaccharide (LPS). In this IFNγ-primed state, not all established NLRP3 activators are specific to NLRP3. We report that nigericin-driven keratinocyte pyroptosis occurs via both NLRP1 and NLRP3, whereas Staphylococcus aureus α-hemolysin (Hla) exclusively and nonredundantly activates NLRP3, even though both require K+ efflux. Furthermore, in the presence of T cells, certain virulent S. aureus strains can cause NLRP3-dependent pyroptotic death in keratinocytes in vitro through the cooperative actions of superantigens (SAgs) and Hla. In summary, our findings establish the strict inducibility and functional relevance of the NLRP3 inflammasome in non-myeloid, epithelial cells in vitro. These results resolve conflicting reports and position keratinocytes as a context-specific, non-hematopoietic cellular model for studying NLRP3 activation in host-microbe interactions at barrier tissues. KEY FINDINGS Additional evidence that NLRP3 is absent in resting, nonstimulated human keratinocytes in vitro IFNγ, but not LPS, is a potent ‘priming’ signal for NLRP3 in human keratinocytes in vitro In IFNγ-primed keratinocytes, S. aureus α-hemolysin (Hla) selectively activates NLRP3, whereas nigericin activates both NLRP1 and NLRP3 in vitro SAg and Hla kill keratinocytes via NLRP3-driven pyroptosis in the presence of T cells in vitro GRAPHICAL ABSTRACT

Summary Aneuploidy—defined as gains and losses of chromosomes—is frequently observed in cancer and has been implicated in promoting tumor progression and metastasis. However, the molecular mechanisms underlying this phenomenon remain poorly understood. By generating new in vitro and in vivo models of aneuploidy, we found that aneuploidy confers remarkable resistance to reactive oxygen species (ROS)-mediated cell death. This is a general consequence of aneuploidy, independent of the specific chromosomes gained or lost. Mechanistically, aneuploidy-induced resistance to cell death results from suppressed Poly(ADP-Ribose) Polymerase 1 (PARP1) in aneuploid cells, which inhibits PARP1-mediated cell death after ROS (parthanatos). We validated aneuploidy-associated PARP1 suppression across 15 cell models and human tumors, with pronounced effects in metastatic tumors. Importantly, decreased PARP1 levels in aneuploid cells promote tumor metastasis and vice versa. Through genome-wide CRISPR screen, a focused CRISPRa screen and functional validation, we identified the transcription factor CCAAT/enhancer-binding protein beta (CEBPB) as a critical mediator of PARP1 downregulation and ROS resistance in aneuploid cells. Furthermore, we found lysosomal dysfunction as the upstream mediator of CEBPB activation in aneuploid cells. We propose that during tumorigenesis, aneuploidy-driven CEBPB activation promotes PARP1 suppression fostering ROS resistance and cancer progression. Highlights Aneuploidy universally confers resistance to oxidative stress independent of p53 status, karyotype and cell lineage through inhibition of PARP1 expression and activity Suppressed PARP1 enhances metastatic potential, while PARP1 restoration suppresses metastatic spread, revealing a novel mechanism linking aneuploidy to cancer progression. PARP1 suppression compromises DNA damage repair and cell death to multiple genotoxic stressors, including reactive oxygen species, alkylating agents, and UV radiation. A genome-wide CRISPR screen and a CRISPRa screen identifies CEBPB as the critical transcription factor mediating PARP1 regulation and ROS resistance. Nuclear CEBPB increases significantly after aneuplodization in experimental systems and in scRNAseq of primary human cancer patients.

Recursive splice sites are rare motifs postulated to facilitate splicing across massive introns and shape isoform diversity, especially for long, brain-expressed genes. The necessity of this unique mechanism remains unsubstantiated, as does the role of recursive splicing (RS) in human disease. From analyses of rare copy number variants (CNVs) from almost one million individuals, we previously identified large, heterozygous deletions eliminating an RS site (RS1) in the first intron of CADM2 that conferred substantial risk for attention deficit hyperactivity disorder (ADHD) and other neurobehavioral traits. CADM2 encodes a neuronally expressed cell adhesion molecule that has repeatedly been associated with ADHD and numerous similar traits. To explore the molecular impact of RS ablation in CADM2 , we used CRISPR to model patient deletions and to target a smaller region (~500 base pairs) containing RS1 in both human induced neurons (iNs) and rats. Transcriptome analyses in unedited iNs provided a catalog of CADM2 transcripts, including novel transcripts that retained RS exons. Intriguingly, ablating RS1 altered the gradient of RNA abundance across the first intron of CADM2 , decreased the level of CADM2 expression, and impacted transcript usage. Decreased CADM2 expression was reflected in reduced exon usage downstream of the RS1 site and global alteration to genes involved in neuronal processes including synapse and axon development. Given the scale of our analyses and the widespread association of CADM2 with neurobehavioral traits, we sought to validate these findings using in vivo models and found that rodent models harboring Cadm2 RS1 deletions exhibited significant changes in relevant behaviors and functional brain connectivity. In summary, our analyses demonstrate a functional role for RS as a noncoding regulatory mechanism in a gene associated with a spectrum of neuropsychiatric and behavioral traits.

Genome-wide association studies (GWAS) have contributed significantly to unraveling the genetic bases of complex diseases such as Parkinson’s disease (PD); yet experimental evidence for causation is often elusive. Here, we hypothesized that non-manifesting carriers of a PD-causing mutation in the LRRK2 gene could express genetic modifiers conferring disease protection. Using a pluripotent stem cell-based model, we showed that dopaminergic neurons derived from these individuals were partially protected from the disease in vitro, and that this protective effect is genetically driven. Whole-exome sequencing identified a previously unreported low-frequency variant in cyclin G-associated kinase (GAK) that was associated with a nearly nine-year delay in age at onset among LRRK2 mutation carriers in a local cohort, although replication in additional cohorts was inconclusive. To rule out inter-cohort heterogeneity, we used CRISPR/Cas9-mediated gene editing to isolate the effect of the mutation. We found that the candidate protective variant prevented neuron loss in vitro along with an improvement of several indicators endocytic-mediated transport. Together, our findings provide mechanistic insights into PD pathogenesis and actionable genetic information for the prognosis of PD patients. One Sentence Summary Investigating genetic protection against Parkinson’s disease in non-manifesting carriers of LRRK2 mutations by CRISPR/Cas9-based genome edition.

ABSTRACT Advances in single-cell sequencing have deepened our understanding of cellular identities. However, because they inherently capture only static snapshots, after which no further observations are possible, we cannot compare past and present profiles within the same cell. Thus, multi-time-point whole-genome profiling at single-cell resolution has been a long-standing goal. Here, we introduce the History Tracing-sequencing (HisTrac-seq) platform, which enzymatically labels genomic DNA adenine to “bookmark” gene regulatory statuses. This first enabled the profiling of transcriptomic and epigenetic states in the mouse brain over a period of two months. Furthermore, extending HisTrac-seq to single-cell multi-omics sequencing, we demonstrated the simultaneous mapping of past and present profiles of the same single cells. Analyzing over 93,000 cells, we discovered unexpected, drastic cell identity transitions on a large scale (“identity jumps”). This phenomenon was previously unobservable with current technologies and revealed a hidden layer of developmental plasticity. HisTrac-seq offers a powerful approach to “temporal multi-omics” for disentangling dynamic biological processes involved in development, plasticity, aging, and disease progression.

ABSTRACT The cytokines interleukin (IL)-22 and IL-17 are secreted by innate and adaptive immune cells to drive “type III” responses that protect against extracellular pathogens, promote mucosal barrier integrity, and foster microbiota homeostasis. However, dysregulation of IL-22 and/or IL-17 contributes to autoimmunity, chronic inflammation, and malignancy. Thus, a deeper understanding of mechanisms regulating type III cytokine production could provide new therapeutic targets for a spectrum of immune-mediated diseases. Toward this goal, we performed a genome-wide CRISPR inhibition (CRISPRi) screen to identify factors that regulate IL-22/IL-17 expression in a murine type III innate lymphoid cell (ILC3) model, MNK3, following stimulation with IL-23 and IL-1b. In addition to previously known regulators of type III cytokines, including IL-23 receptor components IL23R and IL12RB1, the screen identified a large set of new factors that either potentiate or attenuate expression of IL-22 and/or IL-17. A subset of these novel factors was chosen for validation, from which two were selected for further study. The nuclear protein, SON, which binds both DNA and RNA, impaired expression of IL12RB1 at the levels of de novo transcription and RNA processing. The second, MAP4K1 (HPK1), is a serine/threonine kinase that is required for IL-22 but not IL-17 expression. Depletion of MAP4K1 in MNK3 also enhanced expression of the type I cytokine, IFNg, which was co-expressed with IL-17, a phenotype reminiscent of pathogenic Th17 cells. Together, results from the CRISPRi screen broaden our understanding of the factors involved in type III immune responses and offer new targets for modulating IL-22/17 expression.

CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale. However, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference (CRISPRi) screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation, and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying novel candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.