Cell

The skin colonist Staphylococcus epidermidis elicits a potent antibody response that can be redirected against an antigen of interest, but this process relies on genetic engineering.1 Here, by adapting bioorthogonal chemistry methods to conjugate antigens to the cell surface, we make the process of generating a commensal vaccine rapid and efficient. A wide variety of bacteria displaying tetanus toxin fragment C (TTFC) elicit an antibody response when applied to mice topically, indicating that the inductive process is not limited to colonists. Colonization occurs at two different sites, the skin and nostril; by colonizing each with S. epidermidis-TTFC, we show that skin colonization yields a moderate IgG response, while nostril colonization elicits a highly potent systemic IgG response and an exuberant IgA response in the nostrils, lungs, and intestine. Two lines of evidence are consistent with the nasal-associated lymphoid tissue (NALT) as the inductive site for nostril colonization: imaging suggesting robust bacterial translocation, and the induction of commensal-specific B cells following colonization. On the skin, TTFC must be conjugated to live S. epidermidis to elicit an antibody response; in the nostrils, live S. epidermidis-TTFC and S. epidermidis mixed with TTFC are equally potent. Commensal vaccination yields a robust response in pet shop mice, and chemical conjugation facilitates antibody responses to a broad array of antigens, including a whole viral capsid and a rotavirus immunogen. Collectively, these findings provide compelling evidence of a translational path for commensal vaccines.

East Asian populations, representing over 20% of the global population, remain critically underrepresented in human genomic studies, limiting our understanding of population-stratified genetic variation and its implications for health and disease. Here we present the first phase of the Asian Pan-Genome project (APG), comprising 320 nearly complete, fully phased haploid genome assemblies from 160 East Asian individuals. These assemblies achieve unprecedented quality, with an average contig N50 of 144.3 megabase pairs and an average quality value of 64.5. Leveraging these superior assemblies, we reveal previously uncharacterized diversity in human repeatome, including population-stratified patterns in centromere satellites and rDNA arrays. Compared to existing global human genome assemblies, the newly generated genomes supplement 152 million base pairs of novel sequences, 355 gene gains, 18,300 structural variation loci and 26 large euchromatic inversions missing from current human pangenomes. We perform population stratification analyses of structural variations, and further resolve the structural haplotypes of complex genomic regions such as Major Histocompatibility Complex and Survival Motor Neuron loci across global pangenomes, exemplifying tandem-duplicate and inversion-rich complex locus architectures in the human genome, respectively. This resource provides a critical foundation for human genetic studies, especially for East Asian populations, promoting more accurate variant discovery, reducing bias, and ultimately advancing the equity and efficacy of genomic medicine.

Extracellular vesicles (EVs) can deliver RNA and proteins into host cells to manipulate immunity; but how EVs transverse the cell wall is unknown. Using the fungal pathogen Botrytis cinerea that induces cross-kingdom RNA interference in plants, we uncovered that EV-mediated RNA delivery was dependent on cell wall degrading enzymes. Through fluorescence and transmission electron microscopy, and molecular genetic techniques, we demonstrate that EV-associated proteins compromise the plant cell wall, thereby facilitating RNA delivery. Notably, cell wall degrading enzymes are commonly associated with EVs across plant-colonizing bacterial, fungal and oomycete species, indicating a conserved role in EV transport across cell walls. These findings uncover a formerly unknown mechanism by which cell wall degrading enzymes facilitate EVs to transverse the cell wall for cargo delivery in cross-kingdom communication.

Bacteria of the genus Achromatium harbor hundreds of chromosomes that were previously suggested to be genetically diverse. By sequencing multiple regions from individual cells, we demonstrate that chromosomes within a single cell differ in nucleotide and amino acid sequence, reaching levels of divergence well below accepted bacterial species boundaries and below the range associated with homologous recombination. Sequencing of dividing cells further revealed that daughter cells inherit distinct chromosome populations, a mode of inheritance previously associated with sexual reproduction in eukaryotes. Combining subcellular sequencing with high-resolution microscopy, we reconstruct the three-dimensional cellular architecture of Achromatium and show that extensive intracellular heterogeneity arises from the spatial segregation of chromosome populations by the cells internal structure, which limits genome-wide recombination. Our findings establish cellular architecture as a determinant of genome evolution in giant polyploid bacteria and identify spatial genome segregation as a mechanism enabling the maintenance and inheritance of divergent chromosome populations in bacteria.

Mobile genetic elements have profoundly shaped the evolution of bacterial anti-phage immunity, yet much of this defence repertoire remains undiscovered. Antimicrobial-resistance plasmids are well known for disseminating antibiotic-resistance genes, but whether they also serve as reservoirs of previously unrecognised antiviral immunity remains poorly understood. Here, we identify Evangelion, a widespread family of single-gene anti-phage defence systems, through phenotype-guided interrogation of a naturally occurring methicillin-resistant Staphylococcus aureus plasmid. Eva01, the founding member of the family, protects against lytic Silviavirus phages and is built around a conserved DUF4062 core coupled to a highly diversified auxiliary region that is essential for defence activity. Evangelion systems are enriched on antimicrobial-resistance plasmids but are distributed across diverse bacterial hosts and mobile genetic elements, revealing an evolutionarily conserved defence framework that has diversified through horizontal gene transfer and adaptation to distinct phage environments. Genetic, structural and functional analyses support a model in which Eva01 senses intracellular phage replication-associated processes and couples activation of a DUF4062 effector to NAD depletion and abortive infection. Together, our findings define a previously unrecognised family of mobile anti-phage defence systems, establish DUF4062 proteins as a new component of the bacterial anti-phage repertoire, and demonstrate that phenotype-guided interrogation of mobile genetic elements provides a powerful strategy for discovering defence systems beyond the reach of current computational approaches.

Cytokine receptor pairing rules, set by evolution, confine JAK-STAT signaling to a narrow region of a far larger combinatorial space. Of more than 1,200 pairings theoretically possible among the [~]36 JAK-associated human cytokine receptors, only [~]30-40 exist in nature. Using a double-orthogonal platform, we enforced pairings across the full all-by-all receptor matrix and resolved a fine-grained STAT atlas richer than the natural repertoire. Selected non-natural pairings generated emergent T cell states unpredictable from either parental receptor, with pairing orientation encoding signaling specificity. A synthetic IL-21R>IL-2R{beta} pairing, but not its reciprocal, drove a cytotoxic Tc17-like state, whereas natural IL-9R/{gamma}c drove a Tc1 fate despite similar STAT activation, showing that rebalancing quantitative STAT combinatorics can modulate T cell fate. Recombining IL-31R, not expressed in T cells, with STAT-biased receptor partners generated diverse states, several with superior antitumor efficacy. These findings define a non-natural pairing code for engineering synthetic T cell fates.

Eukaryotic DNA transpososomes assemble as nucleoprotein complexes containing multiple identical transposase protomers. We determined the structure of the hyperactive Myotis lucifugus piggyBat transpososome and discovered that it uses an unusual crescent-shaped, asymmetric tetramer to synapse divergent inverted terminal repeats. We found that identical amino-acid sequence motifs adopt distinct roles to mediate two modes of DNA binding: one to perform strand transfer and one, devoid of catalytic activity, that promotes synapsis while simultaneously protecting the transposon from auto-destructive internal cleavage by its active sites. Guided by the observed modularity of the assembly, we engineered an obligate heterodimeric system by identifying mutations that suppress homodimer formation and paired this with specific point mutations that prevent non-targeted integration. By adding to the heterodimer two different TALE domains designed to bind a human genomic safe harbor sequence, we achieved >98% targeted integration at the intended sequence in a plasmid-based assay, validating the viability of heterodimeric transposases for genomic applications.

Mycoviruses are typically transmitted vertically through fungal reproduction or horizontally via hyphal anastomosis, but identical viruses in phylogenetically divergent fungi hint at unknown inter-species transmission mechanisms. Here, we identify fungal extracellular vesicles (EVs) as mediators of cross-genus mycovirus transmission. Using the hypovirulent Botrytis cinerea strain IBc-374 (harboring 16 mycoviruses) as a donor, we show that up to 13 mycoviruses are horizontally transmitted to Sclerotinia sclerotiorum during dual culture or plant co-inoculation, even though these two fungi belong to different genera and are generally considered incapable of hyphal anastomosis. Electron microscopy reveals abundant vesicle structures in IBc-374 hyphae, and nanoparticle tracking analysis showed that the strain secretes >11-fold more EVs than a virus-free strain. RT-PCR detects genomic RNAs of 7 mycoviruses in purified EVs, and the full-length viral genome in EVs was further validated with BcHV5 as example by fluorescence in situ hybridization and RT-PCR. Incubation of protoplast-derived germlings of S. sclerotiorum or B. cinerea with IBc-374 EVs leads to infection by 4-6 donor mycoviruses, demonstrating EV-mediated cross-genus transmission. Injection of mycovirus-carrying EVs into tobacco leaves followed by fungal inoculation also transmits two hypoviruses to both species. Application of IBc-374 hyphal fragment suspension significantly reduces lesion sizes caused by both pathogens on plants, and rescued two pathogens carrying multiple mycoviruses exhibit hypovirulence and impaired growth. Our findings reveal EVs as a cell-free vector that bypasses vegetative incompatibility, providing a mechanistic basis for cross-species viral spread and opening avenues for EV-based virus cocktails to control multiple fungal diseases. IN BRIEFWu et al. discover that fungal extracellular vesicles (EVs) can package and transmit multiple mycoviruses across genera from Botrytis cinerea to Sclerotinia sclerotiorum. EV-mediated delivery overcomes vegetative incompatibility barriers and reduces disease caused by both fungal pathogens, offering a cell-free strategy for mycovirus-based biological control.

Understanding the lineage relationships among individual cells is a key pursuit of modern biology, essential for unraveling the complexities of developmental processes and the adaptive mechanisms of disease progression, particularly in oncology. Retrospective single-cell clonal tracing has emerged as a transformative approach, offering a unique window into the evolutionary trajectories of cancer within clinical samples. While short-read single-cell transcriptomics (scRNA-seq) has revolutionized our ability to map cell states across human tumor atlases, it remains fundamentally limited in its capacity to link these states with high-resolution genomic alterations and the evolutionary trajectories inferred from these natural variants. Integrating somatic mutation discovery with transcriptomic profiles at single-cell resolution often requires separate, costly, and low-throughput genomic assays. Furthermore, existing methods frequently rely on exogenous genetic labeling or are restricted to short-read sequencing, which typically fails to resolve complex genomic rearrangements, large indels, or variations within highly repetitive regions,such as short tandem repeats (STRs), that could serve as potent endogenous clonal markers.

Thousands of genetic variants are associated with autoimmune diseases, but causal variants, their mechanisms, and the pathogenic context in which they act are elusive. Knowledge of pathogenic contexts may enable effective targeted therapies, instead of broad immunosuppressive approaches. First, to focus on the genetics of immune response, we used surface marker CITE-seq data from 1,055,857 peripheral blood mononuclear cells from 356 individuals. We defined genetic associations to 148 surface proteins across eight cell types. We observed a signal in the CD40 locus, implicated in rheumatoid arthritis (RA) and other autoimmune conditions. RA risk variants increased CD40 protein expression by [~]20% on B cells, but with minimal mRNA effects. Second, we deployed base-resolution genome editing, with CRAFT-seq, capturing genomic DNA sequence at the edited site and multimodal phenotypes at single-cell resolution. We defined a single causal allele, rs1883832, within the Kozak motif. Third, we edited this allele, in primary B cells and conducted CRAFTseq to demonstrate trans-effects in >200 genes. These effects were only in the light zone germinal center-like state. Importantly, these trans-effects were not seen in population-scale cohorts of unstimulated B cells. This represents a framework to define disease causal alleles, their cis- and trans-effects. It demonstrates the power of defining causal genetic variation to find trans-effects through editing, which cannot easily be found in population studies.

Comparative transcriptomic studies of neural progenitors implicated in human brain expansion have identified extensive baseline gene expression divergence, yet these differences are weakly enriched for functions relevant to development and disease. This suggests that functionally important regulatory divergence may emerge only under specific developmental signaling conditions. Here, we profiled morphogen-dependent gene expression responses in matched telencephalic neuroepithelial cells (telNECs) from human, chimpanzee, and orangutan at the onset of cortical neurogenesis. Baseline interspecies differences were extensive but functionally diffuse. In contrast, a distinct set of genes exhibited species-divergent responses to morphogen stimulation despite conserved baseline expression. These response genes were strongly enriched for regulators of progenitor proliferation and differentiation, neurodevelopmental disorder risk genes, and loci harboring human-lineage sequence changes. Together, these findings show that developmental signaling exposes a functionally enriched class of regulatory divergence beyond baseline comparisons and provide a framework for identifying evolutionarily relevant gene regulation during human brain development.

Genomic recording enables transient biological signals to be indelibly captured through DNA alterations, creating a permanent record of cellular history retrievable by sequencing. However, current methods are limited by scarce writing space, typically targeting only one or a few amenable genomic sites and requiring large cell populations for signal reconstruction. Here, we establish Repeats for Genomic Recording (RGRs): sequences with up to 400 copies targetable by a single CRISPR guide RNA, readable with a common primer pair, and predicted to have minimal functional impact. We demonstrate that RGRs enable both signal deconvolution in single cells and high-resolution recording in cell populations. Individual RGR sites exhibit distinct response kinetics; thus, combining them improves recording resolution beyond what redundancy alone provides, analogous to diversity reception in wireless communication. We develop a computational pipeline for systematic RGR identification, revealing 15,000 to 25,000 candidates per species across human, mouse, and zebrafish, thereby markedly expanding recording capacity and enabling cell-type-specific applications. Finally, we validate RGRs in live mice by recording long-term immediate early gene activity across the brain following epilepsy induction. This work establishes genomic repeats as a high-capacity platform for single-cell molecular recording in vivo.

IS110 has emerged as a powerful genome-editing tool because it is the smallest RNA-guided system capable of diverse programmable insertions. Naturally existing elements are conventionally modeled as compact[~] 1.5-kb systems comprising a single transposase and a bridge RNA (bRNA). Using high-throughput junction mapping together with large-scale comparative genomics, we redefined the in vivo structural boundaries, growth, and mobilization of IS110 elements. We uncovered a previously unrecognized size continuum extending to[~] 100 kb, driven by progressive local expansion, with expanded loci being widespread across bacterial genomes. Experiments confirmed the activity of natural IS110s both well below and above the size range of previously characterized elements. These large systems preferentially accumulate adaptive cargo, including antimicrobial resistance determinants and heavy-metal detoxification systems, and are strongly enriched for plasmid-derived DNA. Boundary configurations at expanded loci and the range of partial excision intermediates they produce both indicate flexible sequence recognition by IS110, most commonly through half-matches between the bRNA and complementary DNA sequence. This sequence tolerance allows loci to expand with diverse cargo. Together, these findings redefine IS110 from a compact insertion sequence into a dynamic platform that disseminates adaptive cargo.

The human Y chromosome comprises mosaic classes of complex male-specific sequences that remain unclear for their evolution origins, structural variations and pathogenic roles. To address these, we present population-scale analyses of high-quality Y chromosomal sequences of 206 human individuals vs. six ape species, including 160 near complete sequences from the East Asian population, 85 of which are gapless. We uncovered an extensive cryptic diversity of the largest heterochromatin of the human genome on the Y chromosome Yq12, and traced its dual-origin from ancestral centromeric satellites respectively on the Y chromosome and acrocentric autosomes shared with other apes. We showed that such diversity is attributed to the variable and layered expansion of DYZ satellites in the internal array of Yq12, while the Yq12 boundary is marked by conserved inverted arrays. Similarly, the largest Y-linked gene family TSPY can be divided into a highly variable tandem array whose copy number was found to be associated with prostate cancer risks, and an invariable TSPY2 under stronger functional constraints. Within the largest amplicon region AMPL7, we annotated the orientation and copy number per individual of the highly similar amplicons in palindromes, and characterized their recurrent inversion togglings and subsequent microdeletions impacting the male fertility. We further identified population-stratified structural variants in disease-associated genes, including some fixed mutations in TSPY2 and the promoter of DDX3Y in the prevalent Asian haplogroups O1a that are probably associated with elevated prostate cancer risks. Together, our study revealed the mosaic internal structures and evolutionary history of respective complex regions of the human Y chromosome, and provided a foundation for understanding its functional role beyond male determination.

Microbial pathogens require nutrients and water to support their growth and proliferation. Pathogen-mediated resource acquisition is orchestrated by the secretion of virulence factors that have evolved a diversity of forms (from effector proteins to small toxins), but which have converged in function (resource acquisition). A key observable virulence mechanism employed by a large number of microbial pathogens to cause disease is to induce a water-rich niche in the extracellular space (i.e. the apoplast) of their host, known as water-soaked lesions. Given the ubiquity of this virulence mechanism, we asked whether plants respond to pathogen-induced extracellular water to activate immune programs. We find that inducing a water-soaked apoplast induces an atypical transcriptional response, yet results in a functional immune response that restricts pathogen growth as effectively as canonical pattern-triggered immunity. Genetic and functional analyses reveal a role for the immune phytohormone salicylic acid (SA) in mediating apoplastic water-induced immunity (AWII). These results suggest that plants engage in an immune-priming program in response to apoplastic water accumulation, suggesting that the pathogenic niches induced during infection may be perceived as danger signals.

The mammalian cerebral cortex is built from a conserved developmental program, yet exhibits profound species-specific complexity. To decode the regulatory changes driving human brain evolution, we reconstructed and aligned continuous single-cell differentiation trajectories across the developing human, macaque, mouse, and ferret cortices. This comparative framework revealed a fundamental principle of transcriptomic evolution during mammalian cortical development: while stable expression is the mammalian default, genes that diverge strictly shift their allocation to cell differentiation trajectories and developmental timing in tandem. By isolating these coupled regulatory shifts to the human lineage, we revealed that a canonical synaptic gene network uniquely redeployed into early human oligodendrocyte precursor cells (OPCs). Human, chimpanzee, and gorilla cortical organoids confirmed that this neuron-like OPC state is an exclusively human innovation. Spatial transcriptome analysis found that these specialized OPCs engage adjacent neural progenitors (outer radial glia) via synaptic-adhesion signaling during neurogenetic period. These findings demonstrate that this coupled spatiotemporal rewiring establishes novel developmental microenvironments, providing a discrete molecular engine for human cortical evolution.

Reconstructing clonal relationships among human cells is fundamental to understanding development, aging, and disease. Somatic mitochondrial DNA (mtDNA) mutations act as endogenous single-cell barcodes measurable alongside cell-state profiles, but lineage tracing has traditionally focused on high-heteroplasmy variants, which are easier to detect but few and potentially shaped by selection. Whether the more abundant lower-heteroplasmy variants encode bona fide lineage information has not been tested against an independent clonal reference. Using lentiviral barcoding of human hematopoietic cells to establish ground-truth clone identities, we show that after stringent molecule-level error filtering, mutation calls below 10% per-cell heteroplasmy account for roughly half of all lineage-informative calls. Retaining the full heteroplasmy spectrum approximately doubled the clonal-assignment area under the precision-recall curve relative to a >10% cutoff, and single-molecule-supported calls improved recovery when retained collectively. These findings establish lower-heteroplasmy mtDNA mutations as an abundant, bona fide record of clonal history, substantially expanding the clonal resolution attainable in human tissues without genetic engineering.

T cell antigen-specific immunity depends on pairwise interactions between T cell receptors and peptide-MHC, yet isolating the TCR-pMHC pairs that drive productive engagement remains a major obstacle for antigen-specific therapeutics and for decoding TCR specificity. We overcome this by co-encoding TCR and pMHC in a single founder cell, then clonally expanding it inside a semi-permeable capsule so that genetically identical daughter cells engage in trans. T cell activation, rather than binding affinity, is used to sort cells with functional pairs, and a single PCR on the clone's linked genomic library captures both partners. This platform, LINC-seq, recovered known cognate pairs from pooled libraries at up to 95% accuracy and performed simultaneous, library-on-library deep mutational scanning of both partners. Wild-type clonotypes ranked among the top-enriched sequences in complex mixtures, and the screens resolved co-evolutionary epistasis and cross-reactivity rules inaccessible to one-sided mutagenesis. The approach generalizes to any receptor-ligand pair whose trans-engagement drives a reporter.

Transposons were originally described as controlling elements, yet the molecular basis and evolutionary origin of their transcriptional regulatory activities remain elusive. Here, we show that transcriptional regulatory activity is an intrinsic property of transposases (TPases). We demonstrate that the Arabidopsis AtMu1 TPase is sufficient to induce sequence-specific transcriptional activation of cognate copies, revealing a pre-existing cis-regulatory network. We further demonstrate that long-recognized Spm transposon-encoded transcriptional regulator TnpA evolved from an ancestral TPase, linking transcriptional regulators to TPases. Across eukaryotes, we identify multiple domesticated TPase-derived proteins that recurrently lack catalytic residues for transposition, consistent with a one-step regulatory co-option through loss of catalytic activity. Together, our results reveal TPases as autonomous sequence-specific transcriptional regulators and provide a direct evolutionary route from TPases to transcription factors.

Ecosystem resilience in dynamic coastal oceans is conventionally ascribed to functional redundancy, where taxonomically distinct microbes buffer environmental fluctuations through interchangeable metabolic roles. In this study, we reveal a deterministic succession architecture sustained by active non redundancy and temporal metabolic coupling, uncovered via autonomous drone array metatranscriptomic sampling in Daya Bay, China. By resolving the transcriptional landscape into six chronometrically phased modules rather than simple time points, we demonstrate a near total renewal of the active gene pool, with greater than 90% of transcribed clusters showing phase exclusivity within approximatively a two-hour window. This radical functional reshaping exposes a tight, scale dependent taxonomic functional coupling where community composition is a strong, linear predictor of metabolic output, peaking at the genus level (p < 0.0001). Functional continuity is maintained not by redundant generalists, but by a precisely sequential relay of specialists, each optimized for transient microniches with minimal overlap between successive phases. Deep learning structural adjudication of the psbA gene pool further reveals viral orchestration of this temporal coupling, where incoming cyanophages introduce structurally distinct protein variants that sustain photosynthetic electron flow under peak irradiance. These findings redefine coastal microbiome stability as a fine-tuned rapid succession of temporal specialists rather than a redundant backdrop. We propose a fundamental revision of marine ecosystem models, shifting from passive buffering frameworks to deterministic, clock driven architectures, which may prove to be critical for forecasting microbiome responses to accelerating short term climate variability.