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Cheng, Y.

Publications and source records attributed to Cheng, Y..

At least 91 records · Page 5Linked to original sources
bioRxiv2025

Improved Mutation Detection in Duplex Sequencing Data with Sample-Specific Error Profiles

Duplex sequencing enables highly accurate detection of rare somatic mutations, but existing variant callers often rely on protocol-specific heuristics that limit sensitivity, reproducibility, and cross-study comparability. We present DupCaller, a probabilistic variant caller that builds sample-specific error profiles and applies a strand-aware statistical model for mutation detection. Across 50 synthetic datasets, DupCaller identified 1.25-fold more single-base substitutions (SBSs) and 1.41-fold more indels than a state-of-the-art method, while exhibiting equal or better precision. In three duplex-sequenced cell lines treated with aristolochic acid, it recovered expected mutational signatures while detecting 3.5-fold more SBSs and 2.8-fold more indels. In 93 tissue samples-- including neurons, cord blood, sperm, saliva, and blood--DupCaller showed consistent gains, detecting 1.21- to 2.7-fold more mutations. Sensitivity scaled with sample duplication rate, yielding approximately 1.5-fold more mutations under optimal conditions and over 3-fold more in low-duplication samples where other tools falter. These results establish DupCaller as a robust and scalable solution for somatic mutation profiling in duplex sequencing across diverse biological and technical contexts.

bioinformatics↗
bioRxiv2025

BrainBeacon: A Cross-Species Foundation Model for Single-cell Resolved Brain Spatial Transcriptomics

The brains functional complexity emerges from spatiotemporal heterogeneity in molecular, cellular, and circuit organization, shaped by evolution, aging, and disease. Single-cell spatial transcriptomics provides a transformative framework to decode this complexity, resolving cell-type-specific expression patterns, region-specific regulation, evolutionary divergence, and disease-associated disruptions. Despite recent advances in spatial transcriptomics, a unified foundation model capturing cross-species, whole-brain cellular architecture remains lacking. Here, we introduce BrainBeacon, the first cross-species foundation model for whole-brain spatial transcriptomics. Trained on 133 million spatially resolved cells covering a total area of 210194 mm2 from human, macaque, marmoset and mouse brains profiled across five major platforms, BrainBeacon integrates gene expression rankings, spatial cellular organization, and evolutionarily conserved genetic relationships into a unified transformer-based architecture. A two-stage training strategy captures both intra-cellular transcriptional dependencies and inter-cellular spatial interactions, yielding biologically grounded and interpretable representations of brain structure. Subsequently, BrainBeacon enables cross-species alignment of brain cell types and anatomical regions, facilitating integrative analyses of conserved and divergent cellular architectures. Moreover, it uncovers spatial regulatory mechanisms of aging by predicting bidirectional perturbation responses between cells and their local niche. Together, BrainBeacon serves as a foundational cross-species model for whole-brain spatial transcriptomics, enabling virtual brain atlas construction, systematic in silico perturbation studies, and mechanistic insights into brain disease pathogenesis.

bioinformatics↗
bioRxiv2025

Non-disruptive 3D profiling of combinations of epigenetic marks in single cells

Recent advancements in single-cell sequencing and spatial omics technologies have enhanced our understanding of diverse cellular identities, compositions, architectures, and functions. However, the single-cell three-dimensional (3D) organization of the epigenome is still not well understood, due to an absence of spatial single-cell methods that allow high-resolution, locus-specific detection of combinations of epigenetic marks while maintaining the 3D organization of the genome. Here, we develop Epigenetic Proximity Hybridization Reaction (Epi-PHR), a non-disruptive image-based single-cell spatial epigenetic profiling technology. Epi-PHR enables locus-specific and high-resolution in situ detection of combinations of epigenetic marks at hundreds of single gene targets within the same individual cells, while retaining the 3D organization of the genome. Dual-mark Epi-PHR in hippocampus tissue sections revealed region-specific epigenetic profiles. Phased Epi-PHR combined with chromatin tracing simultaneously detects allele-specific epigenetic states and chromatin conformations of a paternally imprinted Meg3 gene cluster in single cells, revealing associations between specific epigenetic mark enrichment and chromatin folding features for the distinct alleles from different parental origins. Surprisingly, we found that H3K9me3 abundance is positively associated with a chromatin domain boundary at the Meg3 locus among maternal copies. We expect Epi-PHR will be broadly applicable in research requiring single-cell spatial epigenetic information, and will help extend our understanding of the combinatorial epigenetic code and its relationship with chromatin organization.

genetics↗
bioRxiv2025

Human placental stem cells induce a novel multiple myeloid cell-driven immunosuppressive program that ameliorates proinflammatory CNS pathology

Despite a growing interest in Amniotic Epithelial Cell (AEC)-based therapies, the immune responses triggered by AEC transplantation in vivo remain poorly characterized. In particular, how direct exposure to AECs within the central nervous system (CNS) shapes the local immune environment is currently unknown. Herein we describe a novel CNS- specific immunoregulatory pathway induced by intracisternal delivery of human AECs. Local immune responses induced by AECs in the brain led to recruitment of immunosuppressive Arginase 1+ (ARG1+) macrophages and a novel population of myeloid-derived suppressor cells with eosinophilic characteristics, which we term Eo- MDSCs. We further demonstrate that Eo-MDSCs produce Maresin 2 (MaR2), a specialized pro-resolving mediator (SPM) involved in the resolution of inflammation. In a mouse model of Multiple Sclerosis (MS), treatment of established disease with AECs induced immunological responses that resulted in reduced numbers of pathogenic macrophages and T helper (TH)17 cells, increased anti-inflammatory T cell subsets, and enhanced myelin phagocytosis, all of which led to functional recovery. These findings suggest that AEC therapy has the potential to target CNS-intrinsic inflammatory processes in MS, providing a strong rationale for translation into the clinic.

neuroscience↗
bioRxiv2025

Cognition-centric brain activity across diverse imaging tasks constrains the representation of mental health in task-evoked brain function

Large-scale neuroimaging studies best powered to investigate mental health biomarkers continue to administer a set of in-scanner tasks that, to date, yield weak brain-mental health associations. Triangulating the source of these weak associations is critical to directing the focus of future work. Using these tasks strong brain-cognition associations as a positive control, we analyzed potential sources of weak brain-mental health associations across five popular fMRI tasks and dozens of task-evoked brain networks. Across tasks and networks, latent mental health factors showed substantially weaker brain associations than cognition despite comparable measurement reliability and greater interindividual variability. Moreover, increasing brain-feature dimensionality improved cognitive but not mental health signal, and task-evoked network profiles were 1.8-2.3 times more homogeneous and 3.5 times lower-dimensional than mental health profiles. Findings implicate a signal evocation bottleneck: common fMRI tasks elicit little interindividual variation in mental health-related brain activity relative to the diversity of mental health itself. Overall, these findings motivate a shift in focus to the development and use of tasks in large-scale studies more explicitly designed to evoke mental health-relevant brain activity. Significance StatementIdentification of robust individual-specific mental health-related brain signals from fMRI remains elusive, limiting the clinical utility of brain imaging. To investigate why, we systematically evaluated brain signals evoked by tasks used by the Human Connectome Project-Young Adult study, which are now commonly included in many large-scale neuroimaging studies. Our findings suggest that, more so than analytical or measurement constraints on detecting these signals, a primary limitation is a failure of these prevalent fMRI tasks to evoke mental health-relevant brain signals in the first place. These findings suggest that progress in psychiatric neuroimaging may require tasks more explicitly designed to evoke mental health-relevant brain activity.

neuroscience↗
bioRxiv2025

The structural basis of cold sensitivity

Thermosensitive TRP ion channels enable somatosensory nerve fibers to detect changes in our thermal environment over a wide physiologic range 1-3. In mammals, the menthol receptor, TRPM8, is activated by temperatures below [~]26{degrees}C and is essential for the perception of cold or chemical cooling agents 4-6. A fascinating, yet still unachieved goal is to elucidate structural mechanisms whereby TRPM8 or other thermosensitive channels are gated by changes in ambient temperature. Recent studies using cryogenic electron microscopy (cryo-EM) have attempted to address this challenging question but are limited by difficulties in visualizing temperature-evoked conformational substates or assessing the energetic landscape governing gating transitions 7,8. Here, we close this gap by using cryo-EM to visualize TRPM8 channels in cellular membranes, where bona fide menthol- and cold-evoked open states are captured. We identify a novel semi-swapped architecture in which interdigitation of channel subunits is substantially rearranged following repositioning of the S6 transmembrane helix and elements of the pore region. By combining this structural analysis with thermodynamic measurements using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we are able to pinpoint the pore and TRP helices as key regions undergoing stimulus-evoked conformational dynamics that drive channel gating. Structural mechanisms associated with activation are validated by comparison of human TRPM8 with the menthol-sensitive, but relatively cold-insensitive avian orthologue. We propose a free energy landscape to explain channel gating by cold or cooling agents.

biophysics↗
bioRxiv2025

Early Radiation Therapy Response Assessment using Multi-scale Photoacoustic Imaging

There is a critical unmet clinical need to identify biomarkers that predict and detect radiotherapy response in cancer. Using the unique capabilities of multi-scale photoacoustic imaging (PAI) for depicting tumour oxygenation and vasculature in vivo, we identified surrogate biomarkers of radiation response in two human breast cancer models (MCF7 and MDA-MB-231), comparing hypofractionated delivery with an ablative single dose scheme. Ex vivo immunohistochemistry results underpinned findings from mesoscopic and multispectral tomographic PAI, performed 24h pre-RT, 24h post-RT, and at endpoint. A denser and more mature vasculature of the MCF7 xenografts afforded an improved response to both RT schemes compared to MDA-MB-231, in terms of overall tumour oxygenation, tumour volume and proliferation. Increased intratumoural blood oxygen saturation and oxygen diffusion pre-RT were associated with improved outcomes and decreased proliferation, expected given the oxygen-enhancement effect in RT. In vivo PAI revealed the differential effect between ablative courses of RT in both models, with the ablative scheme altering the tumour vasculature as early as 24h post-RT, and pruning the looping vessels and total blood volume at endpoint in the more radiosensitive MCF-7 xenografts. An increase in blood oxygen saturation at endpoint was observed only in the MCF7 xenografts treated with hypofractionated RT, confirming the reduced oxygen consumption of damaged tumour cells, indicative of response. Thus, we showed that PAI could capture early RT response and inform on radioresistance, thus demonstrating promise of PAI as an in vivo and future clinical tool to monitor the tumour vascular response to RT.

cancer biology↗
bioRxiv2025

FoldBench: An All-atom Benchmark for Biomolecular Structure Prediction

Accurate prediction of biomolecular complex structures is fundamental for understanding biological processes and rational therapeutic design. Recent advances in deep learning methods, particularly all-atom structure prediction models, have significantly expanded their capabilities to include diverse biomolecular entities, such as proteins, nucleic acids, ligands, and ions. However, comprehensive benchmarks covering multiple interaction types and molecular diversity remain scarce, limiting fair and rigorous assessment of model performance and generalizability. To address this gap, we introduce FoldBench, an extensive benchmark dataset consisting of 1,522 biological assemblies categorized into nine distinct prediction tasks. Our evaluations reveal critical performance dependencies, showing that ligand docking accuracy notably diminishes as ligand similarity to the training set decreases, a pattern similarly observed in protein-protein interaction modeling. Furthermore, antibody-antigen predictions remain particularly challenging, with current methods exhibiting failure rates exceeding 50%. Among evaluated models, AlphaFold 3 consistently demonstrates superior accuracy across the majority of tasks. In summary, our results highlight significant advancements yet reveal persistent limitations within the field, providing crucial insights and benchmarks to inform future model development and refinement.

bioinformatics↗
bioRxiv2025

Scouring the human Hsp70 network uncovers diverse chaperone safeguards buffering TDP-43 toxicity

Cytoplasmic aggregation and concomitant dysfunction of the prion-like, RNA-binding protein TDP-43 underpin several fatal neurodegenerative diseases, including amyotrophic lateral sclerosis. To elucidate endogenous defenses, we systematically scoured the entire human Hsp70 network for buffers of TDP-43 toxicity. We identify 30 J-domain proteins (2 DNAJAs, 10 DNAJBs, 18 DNAJCs), 6 Hsp70s, and 5 nucleotide-exchange factors that mitigate TDP-43 toxicity. Specific chaperones reduce TDP-43 aggregate burden and detoxify diverse synthetic or disease-linked TDP-43 variants. Sequence-activity mapping unveiled unexpected, modular mechanisms of chaperone-mediated protection. Typically, DNAJBs collaborate with Hsp70 to suppress TDP-43 toxicity, whereas DNAJCs act independently. In human cells, specific chaperones increase TDP-43 solubility and enhance viability under proteotoxic stress. Strikingly, spliceosome-associated DNAJC8 and DNAJC17 retain TDP-43 in the nucleus and promote liquid-phase behavior. Thus, we disambiguate a diverse chaperone arsenal embedded in the human proteostasis network that counters TDP-43 toxicity and illuminate mechanistic gateways for therapeutic intervention in TDP-43 proteinopathies.

biochemistry↗
bioRxiv2025

SCRIPT: predicting single-cell long-range cis-regulation based on pretrained graph attention networks

Single-cell cis-regulatory relationships (CRRs) are essential for deciphering transcriptional regulation and understanding the pathogenic mechanisms of disease-associated non-coding variants. Existing computational methods struggle to accurately predict single-cell CRRs due to inadequately integrating causal biological principles and large-scale single-cell data. Here, we present SCRIPT (Single-cell Cis-regulatory Relationship Identifier based on Pre-Trained graph attention networks) that infers single-cell CRRs from transcriptomic and chromatin accessibility data. SCRIPT incorporates two key innovations: graph causal attention networks supported by empirical CRR evidence, and representation learning enhanced through pretraining on atlas-scale single-cell chromatin accessibility data. Validation using cell-type-specific chromatin contact data demonstrates that SCRIPT achieves a mean AUC of 0.9, significantly outperforming state-of-the-art methods (AUC: 0.68). Notably, SCRIPT obtains a threefold improvement in predicting long-range CRRs (>100 Kb) compared to existing methods. Applying SCRIPT to Alzheimers disease and schizophrenia, we establish a framework for prioritizing disease-causing variants and elucidating their functional effects in a cell-type-specific manner. By uncovering molecular genetic mechanisms undetected by existing computational methods, SCRIPT provides a roadmap for advancing genetic diagnosis and target discovery.

bioinformatics↗
bioRxiv2025

A real-time detection and non-destructive warning method for zebrafish body surface anomalies based on improved YOLOv8 framework

The detection of anomalies on fish surfaces is of critical importance for assessing fish health status, preventing fish disease outbreaks, predicting changes in water quality, enhancing fish welfare. The zebrafish (Danio rerio), a key model organism, has been increasingly utilized in various fields, including medicine, genetics and environmental toxicology. This has led to a corresponding increase in demand for intelligent management and detection systems. However, traditional methods of fish disease detection may have irreversible effects on fish, particularly small species, and often fail to meet the precision, non-destructive warning, and real-time requirements for zebrafish detection. To address this issue, this study proposes a novel method based on the YOLOv8 framework, designated ESC-YOLOv8-seg. This method significantly enhances the precision and speed of detecting surface abnormalities on small fish in complex settings by integrating the EMA the SPPELAN and C2f-Faster modules, and adding a detection head tailored for small targets. Furthermore, the integration of positional information and surface features enables the method to achieve real-time monitoring and non-destructive early warning of fish surface abnormalities. The proposed method enhances precision in small target detection and achieves high accuracy in discerning subtle differences among detection targets. In real aquaculture settings, it can reach an average speed of 106 FPS with a detection accuracy of 98%. Although this study has been designed to meet the specific needs of zebrafish scientific research, it is highly generalisable and can be applied to the real-time detection of underwater surface abnormalities in a range of fish species in aquaculture.

bioinformatics↗
bioRxiv2025

A Novel No-Wash (Homogenous) High-Throughput Toxicity Assay on Primary Cells

Mitochondrial membrane potential (MMP) is a critical marker of mitochondrial and, therefore, cellular function. However, current dyes used for MMP measurement possess significant limitations: nonspecific targeting, long incubation times, potential mitochondrial toxicity, poor water solubility, and numerous wash steps. Using a novel fluorescent dye--mitochondrial membrane potential indicator (m-MPI)--in combination with a masking dye, we overcame said limitations and successfully measured MMP changes on different cell lines, as well as four different human primary cells. This no-wash assay has been miniaturized into a 384-well format. It is robust with Z value > 0.5 on both HepG2 and HeLa cells and can be used for high throughput screening. The reagents in the m-MPI homogeneous assay kit are not toxic to cells, as was demonstrated on HepG2 and HeLa cell lines using Dexorgens EnerCount ATP assay. Using this assay, we conducted a mini screening on human hepatocytes against the Library of Pharmacologically Active Compounds (LOPAC) (Sigma). The resulting hits were then confirmed with follow-up studies.

cell biology↗
bioRxiv2025

A unified eIF1A+ luminal cells-centered hypoxic and "cold" tumor microenvironment promotes PCa progression among different subtypes

PCa is a malignancy with high heterogeneity arises from both tumor microenvironment (TME) and histological subtypes. To achieve superior clinical efficacy, it is imperative to identify unified progression drivers within such heterogeneity in PCa, thereby enabling the development of more consistent and effective therapeutic strategies. In this study, we applied imaging mass cytometry to stain 39 proteins on 71 tissues comprising para-cancer, low-grade acinar adenocarcinoma (LgPAC), high-grade PAC (HgPAC), intraductal carcinoma (IDC) and ductal adenocarcinoma (DAC) tissues, obtaining the spatial proteomic landscape of 345,233 single cells. We discovered a hypoxic eIF1A+ luminal epithelial (LE) cluster exhibiting both translational activation and immunosuppressive properties, which was enriched in high-risk PCa including HgPAC, IDC and DAC, and associated which tumor progression and unfavorable prognosis. Additionally, eIF1A+ LE orchestrates the formation of an immunologically "cold" TME, characterized by the low infiltration of anti-cancer immune cells including PD1- T cells CD163- macrophages. Through in vivo validation and investigator-initiated study, we further found that Homoharringtonine, an inhibitor targeting protein translation process, effectively inhibited PCa growth, alleviated hypoxic microenvironment, and enhanced immune cell infiltration. This study is the first to apply spatial proteomics to delineate consistent molecular features across histological subtypes, providing transformative insights for clinical management of PCa.

cancer biology↗
bioRxiv2025

Uncovering the molecular basis of high morphinan product efficiency in opium poppy through Multi-omics integrated analysis with multi-capsules

Exploring the genetic landscape and corresponding regulatory mechanisms influencing morphine and related alkaloid production efficiency in the opium poppy (Papaver Somniferum L.) is important for sustainable medicinal production. However, information regarding the corresponding medicinal properties of poppy subspecies remains elusive, and the correlation between agronomic traits and morphine production remains unclear. Therefore, this study aims to investigate the distinction between two poppy subspecies: Shan Yang (SY) and Lan Tian (LT). Physiological data revealed that SY exhibited greater tolerance to environmental stress and a higher morphine content than LT, especially during the reproductive stage. To investigate genetic and molecular mechanisms underlying different tolerance traits and morphine synthesis efficiencies, a comprehensive comparison of genomic, transcriptomic, and metabolomic data was conducted, showing significantly higher single nucleotide polymorphism (SNP) frequency in SY (23.78%) compared to LT (23.69%). The proportions of SKIP (Skipped Exon) and MIR (Multi Intron Retention) were higher in SY than in LT. SY showed significantly higher gene expression related to benzylisoquinoline alkaloid synthesis, especially those involved in the steps from 1,2 dehydroreticulinium to morphine, narcotoline and noscapine, compared to LT. The upregulated expression of phenylpropanoid biosynthesis-related genes contributes to its more resistant agronomical traits. It plays a synergistic role in alkaloid production. The higher morphine content in SY resulted from an integrated effect controlled at different levels. Summarily, our findings offer new insights into the molecular mechanisms of morphine synthesis and present valuable gene resources for improving poppy cultivars with higher morphine content.

genomics↗
bioRxiv2025

CDK12 condensation in nuclear speckles confers sensitivity to cyclin K molecular glue degraders

Transcriptional machinery often operates in the configuration of biomolecular condensates, presenting emerging opportunities to develop therapies targeting transcriptionally addicted malignancies. In this study, we discovered that cyclin-dependent kinase 12 (CDK12), a key regulator of transcription elongation, formed dynamic liquid-like droplets within nuclear speckles, a process enhanced by high CDK12 expression and CDK12 inhibitor treatment. Recognizing this unique property, we rationally designed ZSQ253, a modular cyclin K molecular glue degrader that coupled binding moieties for both CDK12 and DDB1. By promoting phase separation-driven assembly and subsequent proteasomal degradation of the CDK12-cyclin K complex, ZSQ253 interfered with oncogenic transcription and demonstrated potent antitumor capability. Further structure-guided optimization yielded HQY1428, a derivative with a multi-site gluing effect and improved drug activity. Given the frequent genomic co-amplification of CDK12 and ERBB2 across diverse human cancers, we showed that combining HQY1428 with HER2 inhibitors provided synergistic therapeutic benefits. Collectively, our findings establish CDK12 condensation within nuclear speckles as an exploitable vulnerability, and introduce a novel mechanism of action underlying cyclin K molecular glue degraders.

cancer biology↗
bioRxiv2025

Hemodynamic Characteristics of Acute Hypersensitivity Reaction Induced by PEGylated Nanocomposite in Rats: Mechanism at the Nano-Bio Interface

BackgroundPolyethylene glycol (PEG)-modified nanocomposites may induce acute hypersensitivity reactions (HSR), including complement activation and hypotension, followed by tachyphylaxis with an unknown mechanism. We established a rodent model of acute HSR, and hypothesized that the formation of protein corona with a composition specific to PEGylated nanoparticles induces an acute and transient microvascular occlusion that entrains the hemodynamic effects. MethodsHemodynamic parameters of renal and cremaster vasculature were measured in anesthetized rats using ultrasonic flowmetry and laser speckle contrast imaging, respectively. Proteomic analysis on the hard corona of dextran-coated magnetic nanoparticles (MNP) with and without PEGylation was conducted after incubation of the nanoparticles with plasma from rats. ResultsPEG-MNP iv. induced a temporary reduction by approximately 30 mmHg in arterial pressure, with significant reduction in renal/cremaster blood flow and cardiac output, followed by tachyphylaxis and thrombocytopenia. PEG-MNP, but not pristine MNP, significantly increased renal vascular resistance with a reduction in the calculated cross-sectional area of renal vessels, suggesting microvascular occlusion. In contrast, the vasodilator acetylcholine decreased both blood pressure and vascular resistance before and after administration of PEG-MNP, suggesting an intact endothelium. Complement depletion by cobra venom factor induced a transient reduction in blood flow and prevented PEG-MNP-induced hemodynamic effects, suggesting an important role of complement activation. Proteomic analysis identified much higher complement proteins in the hard corona of PEG-MNP vs. MNP in plasma from rats; preexposure of rats to PEG-MNP or MNP in vivo greatly reduced plasma proteins with high affinity for PEG-MNP. The results suggest that complement depletion may mediate tachyphylaxis in response to the 2nd dose of PEG-MNP. ConclusionPEGylated nanocomposites-induced complement activation in the protein corona may trigger the hemodynamic effects and subsequent pathophysiological responses in HSR of rats. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/642931v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1554185org.highwire.dtl.DTLVardef@40b9d0org.highwire.dtl.DTLVardef@1671c5borg.highwire.dtl.DTLVardef@76dbb5_HPS_FORMAT_FIGEXP M_FIG C_FIG PEGylated magnetic nanoparticles (PEG-MNPs)-induced complement activation and platelet aggregation mediate the systemic increase in resistance of microvessels in rats.

pharmacology and toxicology↗
bioRxiv2025

Sequencing-free whole genome spatial transcriptomics at molecular resolution in intact tissue

Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, compositions, interactions, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution. Leading spatial-capture or spatial-tagging transcriptomics techniques that rely on in-vitro sequencing offer whole-transcriptome coverage, in principle, but at the cost of lower spatial resolution compared to image-based techniques. In contrast, high-performance image-based spatial transcriptomics techniques, which rely on in situ hybridization or in situ sequencing, achieve single-molecule spatial resolution and retain sub-cellular morphologies, but are limited by probe libraries that target only a subset of the transcriptome, typically covering several hundred to a few thousand transcript species. Together, these limitations hinder unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here we develop a new image-based spatial transcriptomics technology termed Reverse-padlock Amplicon Encoding FISH (RAEFISH) with whole-genome level coverage while retaining single-molecule spatial resolution in intact tissues. We demonstrate image-based spatial transcriptomics targeting 23,000 human transcript species or 22,000 mouse transcript species, including nearly the entire protein-coding transcriptome and several thousand long-noncoding RNAs, in single cells in cultures and in tissue sections. Our analyses reveal differential subcellular localizations of diverse transcripts, cell-type-specific and cell-type-invariant tissue zonation dependent transcriptome, and gene expression programs underlying preferential cell-cell interactions. Finally, we further develop our technology for direct spatial readout of gRNAs in an image-based high-content CRISPR screen. Overall, these developments provide the research community with a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNA species in many biomedical contexts.

genetics↗