CRISPR screens have become standard gene discovery platforms in various contexts, including cancer. Yet commonly available CRISPR-Cas9 tools are increasingly recognized as unfit for in vivo investigations in immunocompetent contexts, due to broad immunogenicity of bacterial nucleases and reporters. Here, we show how conventional CRISPR screens in tumor grafts are systematically jeopardized by immunoediting in syngeneic and humanized immunocompetent hosts, resulting in iatrogenic clonal dropouts and ultimately compromising target identification. To resolve this, we present StealTHY, an immunogen-free CRISPR platform compatible with virtually all immunocompetent designs, enabling preservation of clonal architecture and exposing previously concealed cancer vulnerabilities. Among these, we identify the AMH-AMHR2 axis as a formerly unappreciated metastasis target. Thus, with StealTHY, we provide a new resource to expand the applicability of CRISPR screens to immunocompetent models, including humanized tumor grafts, revealing metastasis regulators of therapeutic relevance.

Biomarkers accurately informing prognostic assessment and therapeutic strategy are critical for improving patient outcome in oncology. Here, we apply a whole-genome, tumor-informed circulating tumor DNA (ctDNA) detection approach to address this challenge, leveraging 1,800 variants across 2,994 plasma samples from 431 patients with non-small cell lung cancer (NSCLC) from the TRACERx study. We show that ultrasensitive ctDNA detection below 80 parts per million both pre- and postoperatively is highly prognostic, and combinatorial analysis of the pre- and postoperative ctDNA status identifies an intermediate risk group, improving disease stratification. ctDNA kinetics demonstrate clinical utility during adjuvant therapy, where patients that "clear" ctDNA during adjuvant therapy experience improved outcomes. Moreover, characterization of patterns in postoperative ctDNA kinetics reveals insights into the timing, risk, and anatomical pattern of relapses. By incorporating longitudinal ultrasensitive ctDNA detection, we propose a refined schema for guiding the stratification and treatment recommendations in early stage NSCLC.

Chromatin structure is a key determinant of gene expression in eukaryotes, but it has not been possible to define the structure of cis-regulatory elements at the scale of the proteins that bind them. Here, we generate multidimensional chromosome conformation capture (3C) maps at base-pair resolution using Micro Capture-C ultra (MCCu). This can resolve contacts between individual transcription factor motifs within cis-regulatory elements. Using degron systems, we show that removal of Mediator complex components alters fine-scale promoter structure and that nucleosome depletion plays a key role in transcription factor-driven enhancer-promoter contacts. We observe that chromatin is partitioned into nanoscale domains by nucleosome-depleted regions. This structural conformation is reproduced by chemically specific coarse-grained molecular dynamics simulations of the physicochemical properties of chromatin. Combining MCCu with molecular dynamics simulations and super-resolution microscopy allows us to propose a unified model in which the biophysical properties of chromatin orchestrate contacts between cis-regulatory elements.

During cancer development, mutations promote changes in gene expression that cause transformation. Leukemia associated with aberrant HOXA expression is driven by translocations of nucleoporin genes or KMT2A as well as mutations in NPM1. The mechanistic convergence of these disparate mutations remains elusive. Here, we demonstrate that mutant nucleophosmin 1 (NPM1c) forms nuclear condensates in human cell lines, mouse models, and primary patient samples. We show NPM1c phase separation is necessary and sufficient to recruit NUP98 and KMT2A to condensates. Through extensive mutagenesis and pharmacological destabilization of phase separation, we find that NPM1c condensates are necessary for regulating gene expression, promoting in vivo leukemic expansion, and maintaining the undifferentiated leukemic state. Finally, we show that nucleoporin and KMT2A fusion proteins form condensates that are biophysically indistinguishable from NPM1c condensates. Together, these data define a new condensate that we term the coordinating body (C-body) and establish C-bodies as a therapeutic vulnerability in leukemia.

The traditional process of antibody discovery is limited by inefficiency, high costs, and low success rates. Recent approaches employing artificial intelligence (AI) have been developed to optimize existing antibodies and generate antibody sequences in a target-agnostic manner. In this work, we present MAGE (monoclonal antibody generator), a sequence-based protein language model (PLM) fine-tuned for the task of generating paired human variable heavy- and light-chain antibody sequences against targets of interest. We show that MAGE can generate novel and diverse antibody sequences with experimentally validated binding specificity against SARS-CoV-2, an emerging avian influenza H5N1, and respiratory syncytial virus A (RSV-A). MAGE represents a first-in-class model capable of designing human antibodies against multiple targets with no starting template.

Vision first evolved in the water, where the spectral content of light informs about viewing distance. However, whether and how aquatic visual systems exploit this "fact of physics" remains unknown. Here, we show that zebrafish use "color" information to suppress responses to the visual background. For this, zebrafish divide their intact ancestral cone complement into two opposing systems: PR1/4 ("red/UV cones") versus PR2/3 ("green/blue cones"). Of these, the achromatic PR1 and PR4, which are retained in mammals, are necessary and sufficient for vision. By contrast, the color-opponent PR2 and PR3, which are lost in mammals, are neither necessary nor sufficient for vision. Instead, they form an "auxiliary" system that spectrally suppresses the "core" drive from PR1 and PR4. Our insights challenge the long-held notion that vertebrate cone diversity primarily serves color vision and further hint at terrestrialization, not nocturnalization, as the leading driver for visual circuit reorganization in mammals.

Microbial eukaryotes remain understudied despite their critical ecological importance, with the exception of a few established models. They are often small, difficult to culture, and resistant to standard labeling and imaging techniques. Here, we use ultrastructure expansion microscopy (U-ExM) to carry out high-resolution volumetric imaging of over 200 cultured planktonic eukaryotes across major lineages. By combining U-ExM with pan- and specific immuno-labeling, we reveal microtubule and centrin-containing elements and assign molecular identities to enigmatic cytoskeletal structures observed previously only by electron microscopy. This comprehensive resource provides a basis for understanding cytoskeletal diversity, phenotypic plasticity, and evolutionary dynamics. Moreover, our approach extends to mixed environmental samples, paving the way for environmental cell biology at ultrastructural resolution-a crucial step toward understanding and protecting ecosystems in the face of accelerating biodiversity loss.

The female Aedes aegypti mosquito's remarkable ability to hunt humans and transmit pathogens relies on her unique biology. Here, we present the Aedes aegypti Mosquito Cell Atlas, a comprehensive single-nucleus RNA sequencing dataset of more than 367,000 nuclei from 19 dissected tissues of adult female and male Aedes aegypti, providing cellular-level resolution of mosquito biology. We identify novel cell types and expand our understanding of sensory neuron organization of chemoreceptors across all sensory tissues. Our analysis uncovers male-specific cells and sexually dimorphic gene expression in the antenna and brain. In female mosquitoes, we find that glial cells, rather than neurons, undergo the most extensive transcriptional changes in the brain following blood feeding. Our findings provide insights into the cellular basis of mosquito behavior and sexual dimorphism. The Aedes aegypti Mosquito Cell Atlas resource enables systematic investigation of cell-type-specific expression across all mosquito tissues.

Symbiosis is widespread in nature and plays a fundamental role in organism adaptation and evolution. In nutritional endosymbiosis, host cells accommodate intracellular bacteria and act as a "metabolic factory," requiring extensive metabolic exchanges between host and endosymbiont. To investigate the mechanisms supporting these exchanges, we used the association between the bacterium Sodalis pierantonius and the insect Sitophilus spp. that thrives on an exclusive cereal diet. Volume electron microscopy uncovered that endosymbionts generate complex membranous tubular networks (tubenets) that connect bacteria and drastically increase their exchange surface with the host cytosol. In situ high spatial resolution chemical analysis indicated that tubenets are enriched in carbohydrates, which are the main substrate used by bacteria to generate nutrients for the host. Multiple membranous structures favoring nutrient absorption are described in multicellular organisms. This work demonstrates that bacteria have convergently evolved a similar "biostrategy" that enhances nutrient acquisition by increasing membrane interface.

The second trimester of pregnancy is a pivotal stage in human immune system development. Utilizing single-cell RNA sequencing and T cell receptor sequencing, we profiled 2,868,420 immune cells from 321 samples across 23 organs, including adult tissues as comparators. We identify an extrathymic CD4+ T cell subset mediating TOX2+ precursor cells' transition to mature naive CD4+ T cells. Contrary to the prevailing paradigm of fetal immune quiescence, we uncover widespread memory/activated T cells and tissue-resident memory clones shared across organs, indicating systemic immune activity beyond localized barrier defense. Cell-cell communication and functional assays indicate two tolerance mechanisms that suppress fetal T cell activation: ARG1+ neutrophils and a PTGES3/PTGER4 signaling pathway. We also find that hematopoietic stem cells (HSCs) disperse across multiple organs and show that HSCs from non-canonical hematopoietic organs differentiate into diverse immune lineages. These findings provide insights into human immune system maturation and tolerance in fetuses and adults.

Many species regenerate lost body parts following amputation. Most limb regeneration research has focused on the immediate injury site. Meanwhile, body-wide injury responses remain largely unexplored but may be critical for regeneration. Here, we discovered a role for the sympathetic nervous system in stimulating a body-wide stem cell activation response to amputation that drives enhanced limb regeneration in axolotls. This response is mediated by adrenergic signaling, which coordinates distant cellular activation responses via the α2Α-adrenergic receptor, and local regeneration responses via β-adrenergic receptors. Both α2A- and β-adrenergic signaling act upstream of mTOR signaling. Notably, systemically activated axolotls regenerate limbs faster than naive animals, suggesting a potential selective advantage in environments where injury from cannibalism or predation is common. This work challenges the predominant view that cellular responses underlying regeneration are confined to the injury site and argues instead for body-wide cellular priming as a foundational step that enables localized tissue regrowth.

Detection of DNA is a fundamental strategy for life to recognize non-self or abnormal-self to subsequently trigger the downstream responses. However, the mechanism underlying DNA sensing is incompletely understood. Here, we show that a key neural executioner, sterile alpha and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1), senses double-stranded DNA (dsDNA) to promote cell death. dsDNA-bound and -activated SARM1 to degrade nicotinamide adenine dinucleotide (NAD+) in a sequence-independent manner. SARM1 bound dsDNA via the TIR domain, and lysine residues in the TIR domain contributed to dsDNA binding. In the cellular context, cytosolic dsDNA from dsDNA transfection or chemotherapy treatment was colocalized with SARM1 and activated SARM1 to elicit NAD+ degradation and cell death, which was abrogated by SARM1 knockout or DNA-binding residue mutation. Consistently, SARM1 knockout blocked chemotherapy-induced neuropathy (CIN) in mice. Our results reveal SARM1 as a DNA sensor, which might be targetable for therapeutic interventions.

T cell-mediated tumor killing underlies immunotherapy success. Here, we used long-term in vivo imaging and high-resolution spatial transcriptomics of zebrafish endogenous melanoma, as well as multiplex imaging of human melanoma, to identify domains facilitating the immune response during immunotherapy. We identified cancer regions of antigen presentation and T cell engagement and retention (CRATERs) as pockets at the stroma-melanocyte boundaries of zebrafish and human melanoma. CRATERs are rich in antigen-recognition molecules, harboring the highest density of CD8+ T cells in tumors. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within CRATERs, characteristic of antigen recognition. Following immunostimulatory treatment, CRATERs expanded, becoming the major sites of activated CD8+ T cell accumulation and tumor killing. In humans, elevation in CRATER density in biopsies following immune checkpoint blockade (ICB) therapy correlated with a clinical response to therapy. CRATERs are structures that show active tumor killing and may be useful as a diagnostic indicator for immunotherapy success.

The origins of the early medieval Magyars who appeared in the Carpathian Basin by the end of the 9th century CE remain incompletely understood. Previous archaeogenetic research identified the newcomers as migrants from the Eurasian steppe. However, genome-wide ancient DNA from putative source populations has not been available to test alternative theories of their precise source. We generated genome-wide ancient DNA data for 131 individuals from archaeological sites in the Ural region in northern Eurasia, which are candidates for the source based on historical, linguistic, and archaeological evidence. Our results tightly link the Magyars to people of the early medieval Karayakupovo archaeological horizon on both the European and Asian sides of the southern Urals. The ancestors of the people of the Karayakupovo archaeological horizon were established in the broader Urals by the Late Iron Age, and their descendants persisted in the Volga-Kama region until at least the 14th century.

Progressive multiple sclerosis (PMS), which is characterized by relentless disease progression, lacks effective treatment. While recent studies have highlighted the importance of B cells in driving compartmentalized central nervous system (CNS) inflammation in PMS pathogenesis, current B cell depletion therapies, such as CD20 monoclonal antibodies, face challenges in targeting plasma cells within the CNS. Here, we treated five patients with PMS (one primary PMS and four secondary PMS) with anti-B cell maturation antigen (BCMA) chimeric antigen receptor T (CAR-T) cell therapy in an ongoing phase 1 clinical trial (ClinicalTrials.gov: NCT04561557). Only grade 1 cytokine release syndrome was observed, and all grade ≥3 cytopenias occurred within 40 days post-infusion in all five patients. Meanwhile, we detected plasma cell depletion in CNS compartments, prolonged expansion and relieved exhaustion of CAR-T cells in the cerebrospinal fluid, and attenuation of microglial activation. These findings provided insights into the potential application of anti-BCMA CAR-T therapy for advancing clinical management of PMS.

Sugarcane is a major crop of unclear origins due to its complex polyploid interspecific genome. We analyzed genome ancestries using whole-genome sequence data from 390 representative accessions based on repeated k-mers and chloroplast phylogeny. The results provided evidence that Saccharum officinarum was domesticated in the New Guinea region from the S. robustum wild species and revealed that its genome is a mosaic involving different S. robustum subgroups. We discovered a wild Saccharum contributor to most modern cultivars, likely originating from East Melanesia. We highlighted two early centers of sugarcane diversification associated with human transport, one in continental Asia through hybridization with different S. spontaneum subgroups and one in the Melanesian and Polynesian islands via hybridization with the discovered ancestor and Miscanthus. Finally, we revealed the genome ancestry of modern cultivars, highlighting untapped wild Saccharum diversity as a source of alleles for breeding programs.

Parkinson's disease (PD) has long been considered an appropriate candidate for cell replacement therapy. We generated high-purity dopaminergic progenitors (A9-DPCs) from human embryonic stem cells and evaluated their safety and exploratory efficacy in a single-center, open-label, dose-escalation phase 1/2a trial (NCT05887466) for PD patients. Twelve patients with moderate-to-severe PD received bilateral putamen transplantation of low-dose (3.15 million cells; n = 6) or high-dose (6.30 million cells; n = 6) A9-DPC with immunosuppression. No dose-limiting toxicities or graft-related adverse events were observed. At 12 months, off-medication Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) part III scores and Hoehn and Yahr stage improved, with greater motor improvements in the high-dose group. Dopamine transporter positron emission tomography (PET) imaging showed increased posterior putamen uptake with greater uptake in the high-dose group after transplantation, supporting graft survival. These findings indicate that bilateral transplantation of A9-DPC is safe and may improve parkinsonian motor symptoms in patients with PD.

Rapid remodeling of actin filament (F-actin) networks is essential for the movement and morphogenesis of eukaryotic cells. The conserved actin-binding proteins coronin, cofilin, and actin-interacting protein 1 (AIP1) act in synergy to promote rapid F-actin network disassembly, but the underlying mechanisms have remained elusive. Here, using cryo-electron microscopy (cryo-EM), we uncover the concerted molecular actions of coronin, cofilin, and AIP1 that lead to actin filament aging and severing. We find that the cooperative binding of coronin allosterically promotes inorganic phosphate release from F-actin and induces filament undertwisting, thereby priming the filament for cofilin binding. Cofilin then displaces coronin from the filament via a strand-restricted cooperative binding mechanism. The resulting cofilactin serves as a high-affinity platform for AIP1, which induces severing by acting as a clamp that disrupts inter-subunit filament contacts. In this "molecular squeezing" mechanism, AIP1 and not cofilin is responsible for filament severing. Our work redefines the role of key disassembly factors in actin dynamics.