N Nature Biotechnology · Dec 05, 2025 In vivo gene editing of human hematopoietic stem and progenitor cells using envelope-engineered virus-like particles Engineered virus-like particles (VLPs) are a promising technology for in vivo gene editing of human hematopoietic stem and progenitor cells (HSPCs). Here we design and test two different VLP envelopes for human HSPC editing in vitro and in vivo. The first is an optimized version of the baboon envelope BaEVTR, which efficiently transduces human HSPCs in vitro. We show that the optimized BaEVTR VLP enables in vivo editing of β2 microglobulin in long-term human HSPCs (31% at 8 weeks after dosing) and editing of two hemoglobinopathy-relevant loci,BCL11AandHBG1/2(26% and 7.5%, respectively, at 5 days after dosing), inducing fetal hemoglobin. Our second VLP design uses a CD133-targeted envelope designed to reduce the transduction of mature blood cells and achieves higher in vivo specificity for HSPCs compared to the optimized BaEVTR VLP. As avoiding delivery in filter organs such as the liver would enhance efficiency and safety, we also demonstrate that both VLPs avoid human hepatocytes in a humanized liver model. Gene therapy Genetic vectors Haematopoietic stem cells biology mouse experiments
N Nature Biotechnology · Dec 03, 2025 Mapping single-cell diploid chromatin fiber architectures using DAF-seq Gene regulation is orchestrated by the co-binding of proteins along chromosome-length chromatin fibers within single cells, yet the heterogeneity of this occupancy between haplotypes and cells remains poorly resolved in diploid organisms. Here we present Deaminase-Assisted single-molecule chromatin Fiber sequencing (DAF-seq), which enables single-molecule footprinting at near-nucleotide resolution while synchronously profiling single-molecule chromatin states and DNA sequence. DAF-seq illuminates cooperative protein occupancy at individual regulatory elements and resolves the functional impact of somatic variants and rare chromatin epialleles. Single-cell DAF-seq (scDAF-seq) generates chromosome-length protein co-occupancy maps across 99% of each individual cell’s mappable genome. scDAF-seq uncovers extensive chromatin plasticity both within and between single diploid cells, with chromatin actuation diverging by 61% between haplotypes within a cell, and 63% between cells. Moreover, we find that regulatory elements are preferentially co-actuated along the same fiber in a distance-dependent manner that mirrors cohesin-mediated loops. Overall, DAF-seq enables the characterization of protein occupancy across entire chromosomes with single-nucleotide, single-molecule, single-haplotype and single-cell precision. Epigenomics Genomics Sequencing biology
N Nature Biotechnology · Dec 03, 2025 Standardized metrics for assessment and reproducibility of imaging-based spatial transcriptomics datasets Spatial transcriptomics lacks standardized metrics for evaluating imaging-based in situ hybridization technologies across sites. In this study, we generated the Spatial Touchstone (ST) dataset from six tissue types across several global sites with centralized sectioning, analyzed on both Xenium and CosMx platforms. These platforms were selected for their widespread use and distinct chemistries. We assessed reproducibility, sensitivity, dynamic ranges, signal-to-noise ratio, false discovery rates, cell type annotation and congruence with single-cell profiling. This study offers ST standardized operating procedures (STSOPs) and an open-source software, SpatialQM, enabling evaluation of samples across all technical metrics and direct imputation of cell annotations. The generated imaging-based spatial transcriptomics data repository comprises 254 spatial profiles, incorporating both public and newly generated ST datasets in a web-based application, which enables analysis and comparison of user data against an extensive collection of imaging-based datasets. Finally, we establish best practices and metrics to evaluate and integrate imaging-based multi-omics data from single cells into spatial transcriptomics to spatial proteomics. Bioinformatics Quality control Standards Transcriptomics other
N Nature Biotechnology · Nov 26, 2025 All-optical visualization of specific molecules in the ultrastructural context of brain tissue Understanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the three-dimensional nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy visualize either specific labels or anatomical ultrastructure but combining molecular specificity with anatomical context is challenging. Here we present pan-expansion microscopy of tissue (pan-ExM-t), an all-optical imaging method that combines ~16–24-fold linear expansion with fluorescent pan-stainings of proteins and lipids (providing electron microscopy-like ultrastructural context) and immunolabeling (for molecular imaging). We demonstrate the versatility of this approach by imaging synaptic and cell-specific antibodies in the ultrastructural three-dimensional context of presynaptic and postsynaptic densities, neuropil nanoarchitecture and cellular organelles in dissociated neuron cultures, and mouse brain tissue sections. Furthermore, we demonstrate tracing of neuronal circuitry from pan-ExM-t image volumes, suggesting that any laboratory with access to a confocal microscope can now localize specific molecules within nanoscale cellular and circuit contexts. Cellular neuroscience Super-resolution microscopy biology mouse experiments
N Nature Biotechnology · Nov 26, 2025 Antimicrobial peptide delivery to lung as peptibody mRNA in anti-inflammatory lipids treats multidrug-resistant bacterial pneumonia The efficacy of antimicrobial peptides (AMPs) is limited by challenges of delivery and potency. We enhance AMP performance in the lung by converting AMPs to a peptibody format that fuses AMPs with fragment crystallizable domains to activate innate immunity and cathelin domains for infection-responsive activation, with their mRNA constructs delivered by anti-inflammatory lipid nanoparticles. The highest-scoring design outperforms antibiotic therapy approved by the US Food and Drug Administration in multidrug-resistant pneumonia models, eradicating representative MDR bacteria while mitigating inflammation. Drug delivery Nanomedicine biology
N Nature Biotechnology · Nov 25, 2025 Multiplexed profiling of transcriptional regulators in plant cells Transcriptional regulators play key roles in plant growth, development and environmental responses; however, understanding how their regulatory activity is encoded at the protein level has been hindered by a lack of multiplexed large-scale methods to characterize protein libraries in planta. Here we present enrichment of nucleartrans-elements reporter assay in plants with sequencing (ENTRAP-seq), a high-throughput method that introduces protein-coding libraries into plant cells to drive a nuclear magnetic sorting-based reporter, enabling multiplexed measurement of regulatory activity from thousands of protein variants. Using ENTRAP-seq and machine learning, we screen 1,495 plant viruses and identify hundreds of putative transcriptional regulatory domains found in structural proteins and enzymes not associated with gene regulation. In addition, we combine ENTRAP-seq with machine-guided design to engineer the activity of a plant transcription factor in a semirational fashion. Our findings demonstrate how scalable protein function assays deployed in planta will enable the characterization of natural and synthetic coding diversity in plants. Assay systems Functional genomics Molecular engineering in plants Plant molecular biology Transcriptional regulatory elements biology
N Nature Biotechnology · Nov 21, 2025 Programmable initiation of mRNA translation bytrans-RNA Several approaches exist to silence genes, but few tools are available to activate individual mRNAs for translation inside cells. Guiding ribosomes to specific start codons without altering the original sequence remains a formidable task. Here we design cappedtrans-RNAs capable of directing ribosomes to specific initiation sites on individual mRNAs when thetrans-cap is positioned near the target start codon. Structural and biochemical data suggest that the cappedtrans-RNA facilitates ribosome loading and scanning on the target mRNA through a synergistic mechanism involving alternative cap recognition. Thetrans-RNA also acts independently of the cap on the target mRNA, enabling translation of circular RNAs lacking internal ribosome entry sites. We applytrans-RNAs in vivo to achieve programmable alternative translation of endogenous genes in mouse liver. Finally, we provide the evidence for the existence of natural transcripts that, similarly to exogenoustrans-RNAs, activate translation of endogenous mRNAs. Ribosome RNA biology mouse experiments
N Nature Biotechnology · Nov 17, 2025 Extensive restoration of forelimb function in primates with spinal cord injury by neural stem cell transplantation Research on cell therapy for spinal cord injury has yet to achieve sufficient functional recovery. Previous studies in the field grafted oligodendrocyte progenitors, nonspinal neural stem cells or primary spinal neural progenitors. Here we sought to improve functional outcomes by grafting clinically compatible spinal cord neural stem cells derived from human embryonic stem cells (H9-scNSCs). H9-scNSCs significantly improved functional outcomes on a skilled hand task 9.2-fold (P= 2.5 × 10−27) in hemisected subjects compared with lesioned controls, achieving a fine object retrieval success of 53.4 ± 19.2%, and 2.9-fold (P= 6.3 × 10−8) superior to controls in hemicontused subjects. Recovery correlated with rehabilitation effort. Grafts extended up to hundreds of thousands of new axons into host circuits up to 39 mm below the injury, forming synapses with host circuitry. Lesion fill was substantially higher and differentiated cell-fate distributions were much closer to that of the normal spinal cord than in previous studies using primary spinal cord cells, likely enabling the observed superior functional outcomes. Embryonic stem cells Neural stem cells Spinal cord injury biology mouse experiments
N Nature Biotechnology · Nov 17, 2025 Fecal exfoliome sequencing captures immune dynamics of the healthy and inflamed gut Metagenomic sequencing and metabolomics of fecal matter have revealed the impact of the gut microbiome on health and disease. In addition to microbiota, feces also contain shed or exfoliated host epithelial, secretory and immune cells, but RNA profiling of these cells is challenging owing to degradation and cross-contamination. Here we introduce exfoliome sequencing (Foli-seq) to profile fecal exfoliated eukaryotic messenger RNAs (feRNAs) originating from the upper and lower gastrointestinal regions and show that this ‘fecal exfoliome’ harbors stable RNAs that reflect intestinal and immune function. By selectively amplifying targeted transcripts, Foli-seq demonstrates robust, accurate, sensitive and quantitative measurement of feRNAs. In murine colitis models, feRNA reveals temporal processes of epithelial damage, immune response and intestinal recovery specific to different types of gut inflammation. Simultaneous exfoliome and microbiome profiling uncovers a dense host–microbe interaction network. Moreover, we demonstrate stratification of patients with inflammatory bowel disease into subgroups that correlate with disease severity. Fecal Foli-seq is a noninvasive strategy to longitudinally study the gut and profile its health. Gene expression analysis Gene expression profiling Inflammation biology mouse experiments
N Nature Biotechnology · Nov 13, 2025 Isotropic, aberration-corrected light sheet microscopy for rapid high-resolution imaging of cleared tissue Light-sheet microscopy is ideal for imaging large and cleared tissues, but achieving a high isotropic resolution for a centimeter-sized sample is limited by slow and often aberrated, axially scanned light sheets. Here, we introduce a compact, high-speed light-sheet fluorescence microscope achieving 850 nm isotropic resolution across cleared samples up to 1 cm³ and refractive indices ranging from 1.33 to 1.56. Using off-the-shelf optics, we combine an air objective and a meniscus lens with an axially swept light sheet to achieve diffraction-limited resolution and aberration correction. The effective field of view is increased by twofold by correcting the field curvature of the light sheet using a concave mirror in the remote focusing unit. Adapting the light sheet’s motion with a closed-loop feedback enhances the imaging speed by tenfold, reaching 100 frames per second while maintaining resolution and field of view. We benchmark the system performance across scales, from subcellular structure up to centimeter scale, using various clearing methods. 3-D reconstruction Fluorescence imaging Light-sheet microscopy Neuroscience other
N Nature Biotechnology · Nov 11, 2025 A parts list of promoters and gRNA scaffolds for mammalian genome engineering and molecular recording A standardized ‘parts list’ of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in human and mouse cell lines, including hundreds with greater activity than commonly used sequences. Saturation mutagenesis screens identify tolerated sequence variants that further enhance sequence diversity. In an application to molecular recording, we design a ‘ten key’ array that, in mammalian cells, achieves balanced activity of pegRNAs as predicted by the activity of the component parts. The data reported here will aid the design of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity for applications in multiplexed perturbation, biological recorders and complex genetic circuits. Functional genomics Genomics biology mouse experiments
N Nature Biotechnology · Nov 11, 2025 Multimodal learning enables chat-based exploration of single-cell data Single-cell sequencing characterizes biological samples at unprecedented scale and detail, but data interpretation remains challenging. Here, we present CellWhisperer, an artificial intelligence (AI) model and software tool for chat-based interrogation of gene expression. We establish a multimodal embedding of transcriptomes and their textual annotations, using contrastive learning on 1 million RNA sequencing profiles with AI-curated descriptions. This embedding informs a large language model that answers user-provided questions about cells and genes in natural-language chats. We benchmark CellWhisperer’s performance for zero-shot prediction of cell types and other biological annotations and demonstrate its use for biological discovery in a meta-analysis of human embryonic development. We integrate a CellWhisperer chat box with the CELLxGENE browser, allowing users to interactively explore gene expression through a combined graphical and chat interface. In summary, CellWhisperer leverages large community-scale data repositories to connect transcriptomes and text, thereby enabling interactive exploration of single-cell RNA-sequencing data with natural-language chats. Gene regulation in immune cells Machine learning Preclinical research Software Transcriptomics biology
N Nature Biotechnology · Nov 10, 2025 Clearance of intracranial debris by ultrasound reduces inflammation and improves outcomes in hemorrhagic stroke models Impaired clearance of neurotoxic debris in the brain exacerbates neurologic disease and presents a promising therapeutic target. Pharmacologic therapies can enhance meningeal lymphatic clearance in preclinical models but may be limited by systemic toxicities or invasive administration. Here we report a low-intensity, focused ultrasound protocol that noninvasively clears pathogenic substances from the cerebrospinal fluid and brain interstitium in mice. Using two models of hemorrhagic stroke, we demonstrate that this protocol clears the cerebrospinal fluid and interstitium of blood cells, which accumulate in the deep cervical lymph nodes via meningeal lymphatics. The protocol directly modulates molecular processes, including mechanosensitive channels, to shift microglial phenotypes and astrocytic aquaporin localization to reduce neuroinflammation and neurocytotoxicity. In the intracerebral hemorrhage model, it improves behavioral outcomes and increases survival with greater efficacy than a pharmacologic benchmark. The protocol satisfies Food and Drug Administration safety guidelines, supporting clinical translatability. If demonstrated effective clinically, it may provide therapeutic benefit not only in hemorrhagic stroke but also in other neurologic disorders that involve impaired debris clearance. Biomedical engineering Stroke biology mouse experiments
N Nature Biotechnology · Nov 07, 2025 RNA stability enhancers for durable base-modified mRNA therapeutics The limited stability of mRNA in vivo remains a major challenge for vaccines and therapeutics. While alternative RNA formats such as circular RNA or self-amplifying RNA offer greater durability, these modalities often suffer from low translation, modification incompatibility and difficult manufacturing. To overcome these limitations, we screen 196,277 viral sequences and identify eleven elements that strongly enhance mRNA stability and translation. Mechanistically, they recruit TENT4 to extend the poly(A) tail, preventing deadenylation. Five of them are compatible withN1-methylpseudouridine, which improves mRNA efficacy and reduces immunogenicity. An element named A7 demonstrates particularly robust performance across cell types, delivery methods, modifications and coding sequences, making linear mRNA as stable as circular RNA while achieving higher translation efficiency. In mouse liver, A7-containing linear mRNA exhibits substantially higher protein levels than circular RNA, with sustained expression lasting for over 2 weeks. These RNA stability enhancers enable robust linear mRNA platforms that combine high and durable expression, low immunogenicity and simple manufacturing. Molecular biology Nucleic-acid therapeutics biology mouse experiments
N Nature Biotechnology · Nov 06, 2025 CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci Existing methods to visualize dynamic changes in the three-dimensional genome, promoter−enhancer interactions and the influence of epigenetic modifications in non-repetitive loci are limited. Here we introduce CRISPR PRO-LiveFISH (Pooled gRNAs with Orthogonal bases LiveFISH), which combines orthogonal bases from expanded genetic alphabet technology and rational single guide RNA (sgRNA) design to efficiently label multiple non-repetitive loci in living cells. The optimized method allows simultaneous imaging of up to six genomic loci and uses as few as 10 sgRNAs for non-repetitive loci imaging without signal amplification. We demonstrate the method in diverse cell types, including primary cells, and apply it to reveal enhancer−promoter dynamics and a correlation between genomic dynamics and epigenetic states. We also show thatPCDHα−enhancer interactions may persist despite spatial mobility and that BRD4 maintains super-enhancer contacts regulatingMYConcogene expression in cancer cells. CRISPR PRO-LiveFISH can be applied to diverse studies of chromatin dynamics and genome organization in living cells. Biotechnology Cellular imaging Genetics biology
N Nature Biotechnology · Nov 06, 2025 Site-specific DNA insertion into the human genome with engineered recombinases Insertions of large DNA sequences into the genome are broadly enabling for research and therapeutic applications. Large serine recombinases (LSRs) can mediate direct, site-specific genomic integration of multi-kilobase DNA sequences without a pre-installed landing pad, albeit with low insertion rates and high off-target activity. Here we present an engineering roadmap for jointly optimizing their DNA recombination efficiency and specificity. We combine directed evolution, structural analysis and computational models to rapidly identify additive mutational combinations. We further enhance performance through donor DNA optimization and dCas9 fusions, enabling simultaneous target and donor recruitment. Our top engineered LSR variants, superDn29−dCas9, goldDn29−dCas9 and hifiDn29−dCas9, achieve up to 53% integration efficiency and 97% genome-wide specificity at an endogenous human locus and effectively integrate large DNA cargoes up to 12 kb for stable expression in non-dividing cells, stem cells and primary human T cells. Rational engineering of DNA recombinases enables precise and efficient single-step genome insertion for diverse applications across gene and cell therapies. DNA recombination Gene therapy Protein design biology
