Biology subjects

Yu, Y.

Publications and source records attributed to Yu, Y..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Unraveling the Transcriptional Landscape within a Minimized Bacterium via Comparative Analysis

Stochastic nature of gene expression leads to the complex formation of the bacterial transcriptome and proteome. In contrast to typical transcriptome studies, we employ a near wild-type, Syn1.0, of the naturally genome-reduced Mycoplasmas, and the dramatically further genome-reduced JCVI-syn3A thus avoiding additional contributions from many non-essential cellular functions. To aid in profiling the transcriptional landscape within these bacteria, we present a bioinformatic analysis of the genetic sequence motifs implicated in modulating the stochastic gene expression events, coupled with genome-wide short-read (Illumina) and long-read (Oxford Nanopore Technologies and Pacfic Biosciences) RNA sequencing. The bioinformatic analysis coupled with information from structural studies assigns strengths of the Shine-Dalgarno signatures and identifies both transcription initiation and termination sites, leading to predictions of RNA isoforms in Syn1.0 (and related organisms). The long-read and short-read RNA sequencing characterized the predicted transcriptional activity, and the long-read methods provide direct insight into the RNA isoform complexity within Syn1.0. Comparison of the RNA sequencing results with that of the bioinformatic analysis highlights the inability of bioinformatics alone to capture the results of bacterial transcription without including effects of RNA degradation. This study emphasizes the need for comparative analysis and potential dangers of genome reduction, exemplified through the discovery of altered gene expression patterns of JCVI-syn1.0 and JCVI-syn3A, achieved via the union of our transcriptome study with their proteomics data. Analysis of the transcriptomics data sets through a Jupyter notebook allows any genomic region to be easily examined. Table of Content Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/681674v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@78a9e0org.highwire.dtl.DTLVardef@1d8c9fcorg.highwire.dtl.DTLVardef@1b4e129org.highwire.dtl.DTLVardef@2a8c3f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Castl: Robust Identification of Spatially Variable Genes in Spatial Transcriptomics via an Ensemble-based Framework

Spatially variable genes (SVGs) are essential for elucidating tissue organization within spatially resolved transcriptomics. While a number of computational methods have been developed for SVG identification, their reliance on algorithm-specific assumptions, such as predefined kernel functions or spatial neighborhood graphs, often results in substantial variability in sensitivity and inflated false discovery rates (FDRs) across heterogeneous datasets. To address this challenge, we here develop Castl, an ensemble-based framework for SVG identification that integrates multiple detection methods through statistically designed aggregation modules. Comprehensive evaluations on both simulated and real-world data demonstrate that Castl consistently identifies biologically meaningful spatial expression patterns, mitigates method-specific biases and effectively controls FDRs across various biological contexts, resolutions, and spatial technologies. This flexible, assumption-free framework offers a robust and standardized foundation for spatially informed feature discovery in complex biological systems.

bioinformatics↗

bioRxiv2025

Chronic Ethanol Drinking Alters Medial Prefrontal Cortex and Nucleus Accumbens Astrocyte Translatome and Extracellular Matrix Glycosaminoglycans

Alcohol Use Disorder is a leading preventable cause of morbidity and mortality, yet knowledge of mechanisms driving ethanol-related neuroplasticity remains incomplete. While research has traditionally focused on neuronal signaling, emerging evidence implicates astrocytes in addiction-related adaptations. Here, we investigated the astrocyte-specific molecular consequences of chronic ethanol consumption in the prefrontal cortex and nucleus accumbens, two brain regions critical for executive control and reward processing. Using Translating Ribosome Affinity Purification RNA-seq and bulk RNA-seq in Aldh1l1-EGFP/Rpl10a mice, expressing an EGFP tag on astrocyte ribosomes, we identified hundreds of differentially translated astrocytic genes following chronic continuous two-bottle choice ethanol drinking. Sex-specific analyses revealed greater astrocytic changes in the female PFC and male NAc. Pathway enrichment highlighted extracellular matrix remodeling, synaptic signaling, mitochondrial function, and immune-related pathways. Analyses of individual drinking levels further demonstrated distinct correlations between ethanol intake and astrocytic translation. The major components of the brain extracellular matrix are chondroitin sulfate proteoglycans, produced primarily by astrocytes and covalently bound to chondroitin sulfate glycosaminoglycan chains. Complementary mass spectrometry/liquid chromatography analyses of chondroitin sulfate, heparan sulfate, and hyaluronic acid glycosaminoglycan disaccharides revealed ethanol-induced alterations in chondroitin sulfate glycosaminoglycan sulfation patterns, with additional baseline differences identified between selectively bred high- and low-ethanol preference lines. Together, these findings indicate that astrocytes undergo profound sex- and region-specific adaptations to chronic ethanol, implicating extracellular matrix and glycosaminoglycan remodeling as key risk-factors for and mediators of chronic ethanol-related neuroplasticity.

neuroscience↗

bioRxiv2025

Contralateral-Ipsilateral Segregation as a Conserved Principle of the Visual Corpus Callosum

How the brain maintains distinct information streams within dense white matter is a fundamental question. We investigated whether the visual corpus callosum transmits information via segregated "parallel wires" or mixed pathways. This distinction is critical: a mixed architecture would render the signals origin ambiguous, whereas a segregated arrangement implies that spatial position tracks the direction of information flow. Using high-field fMRI and Bayesian modeling in humans, we demonstrate a segregated architecture featuring distinct contralateral and ipsilateral channels. This functional segregation mirrors a precise anatomical arrangement in mice, where dual-color viral tracing and light-sheet microscopy reveal that callosal axons remain spatially segregated in distinct laminae after crossing the midline. Our findings establish a conserved "parallel wires" principle of callosal organization, providing a new framework to decode directional information flow and assess pathway-specific damage in neurological disease.

neuroscience↗

bioRxiv2025

Computational modeling of optogenetic control in human atrial tissue: The role of fibrosis type and illumination strategy

Atrial fibrosis promotes atrial fibrillation by stabilizing spiral waves, with focal fibrosis causing strong anchoring that resists termination. Optogenetics offers a low-energy alternative for rhythm control, but its efficacy in human atrial tissue remains under investigation. Here, we developed a two-dimensional computational model of human atrial tissue expressing the light-sensitive protein GtACR1, coupled with fibroblasts, to simulate spiral wave dynamics under varying degrees (0%-100%) and types (focal vs. diffuse) of fibrosis. We examined the effects of subthreshold illumination area (0%-100%) and spatial intensity profiles (uniform, parabolic, linear, exponential) on termination efficiency, and tested low-intensity periodic stimulation for wave drift induction. Results show that the minimum light area required for termination increases with fibrosis degree. At 25%-50% fibrosis, focal substrates require larger illumination areas than diffuse ones, confirming stronger anchoring. Crucially, the relationship between illumination area and termination time is non-monotonic. An "optimal light range" exists: smaller areas fail to eliminate the wave, while excessively large areas suppress wavefront collisions, prolonging termination. In diffuse fibrosis, low-intensity periodic illumination successfully induces drift and boundary collision, enabling self-termination. In contrast, in focal fibrosis, the spiral wave core remains anchored, and drift cannot be initiated by modulating stimulation frequency or intensity alone. Our findings demonstrate that fibrosis type and extent critically influence optogenetic control efficacy. The existence of an optimal illumination strategy highlights the need for spatially tailored interventions. These results provide a mechanistic basis for developing individualized, low-energy optogenetic defibrillation protocols. Author summaryAtrial fibrillation, a common heart rhythm disorder, is often sustained by rotating electrical waves in the heart muscle. Scarring (fibrosis) in the atria can anchor these waves, making them harder to eliminate. Optogenetics -- a technique that uses light to control genetically modified heart cells--offers a promising low-energy approach to stop these dangerous rhythms. However, how different patterns of scarring affect the success of optical interventions remains unclear. In this study, we developed a computational model of human atrial tissue expressing a light-sensitive protein (GtACR1) to investigate how two major types of fibrosis--localized "focal" scars versus widespread "diffuse" scarring -- influence the effectiveness of light-based rhythm control. We found that focal fibrosis strongly anchors spiral waves, requiring larger illuminated areas for termination, while diffuse fibrosis allows more efficient disruption with smaller light zones. Crucially, we discovered an "optimal light range": too little light fails to stop the wave, but too much can paradoxically prolong termination by suppressing wavefront collisions. Furthermore, we show that low-intensity periodic light can induce drift and self-termination in diffuse fibrotic tissue, but fails in focal substrates due to structural anchoring. Our findings highlight that one-size-fits-all optical strategies are suboptimal, and that personalized illumination protocols--tailored to individual fibrosis patterns--could enable safer, lower-energy defibrillation in the future.

physiology↗

bioRxiv2025

A molecular stabiliser of an inhibitory eIF2B-eIF2(αP) complex activates the Integrated Stress Response

Eukaryotic initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF), promotes protein synthesis by charging translation initiation factor 2 (eIF2) with GTP. Stress-induced phosphorylation of eIF2 on its -subunit [eIF2(P)] inhibits this reaction triggering a protective Integrated Stress Response (ISR). A DNA-encoded chemical library (DEL) screen for modulators of eIF2B, led to the identification of a chemical series that inactivates eIF2B, stimulating the ISR. Cryo-EM of compound-bound eIF2B revealed a conformational switch to the inactive state engaged by eIF2(P). In cells, compound activity was sensitive to eIF2s phosphorylation state and to a competing eIF2B ligand (ISRIB) that activates the GEF allosterically. These findings mark the discovery of a first-in-class drug-like allosteric inhibitor of eIF2B, an ISR activator (ISRAC), paving the way to explore the therapeutic potential of eIF2B-directed ISR activation.

molecular biology↗

bioRxiv2025

Vision dominates sound in mediating classic cue-induced microsaccadic eye movement modulations in rhesus macaque monkeys

Visual onsets in the environment cause a short-latency reduction in saccade generation likelihood. Concomitant with that, the metrics of the saccades that do happen near the time of saccadic inhibition reflect the landscape of stimulus-driven neural responses in topographically organized brain maps. While certain aspects of saccadic inhibition have already been explored from a neurophysiological perspective, several questions remain, including which sensory-motor pathways propagate visual input information to the final oculomotor control circuitry with such short latencies. Here, motivated by evidence of a dissociation between temporal and spatial aspects of saccadic inhibition, we devised behavioral paradigms experimentally manipulating either aspect. Rhesus macaque monkeys maintained gaze fixation, and we characterized visually-driven microsaccadic inhibition with and without paired auditory pulses. In one condition, the sound pulses were spatially-nonlateralized, thus only affecting the temporal processes associated with microsaccadic inhibition; in another, the pulses were spatially-lateralized, affecting both the temporal and spatial components. We found that sound pulses alone barely affected microsaccade rates. However, when paired with visual stimuli, they accelerated the timing of visually-driven microsaccadic inhibition by [~]14-18 ms. In terms of microsaccade directions, spatially-nonlateralized sounds interfered with, but did not eliminate, visually-driven direction modulations. Surprisingly, lateralized sounds had a much weaker effect on microsaccade direction modulations, even when the sound source was in the opposite hemifield as the appearing visual stimulus. We conclude that vision dominates sound in mediating classic microsaccadic inhibition, and we argue that our paradigms are ideal for neurophysiological investigations of the parallel sensory-motor pathways contributing to such inhibition. SignificanceCoordination of internal oculomotor eye movement plans with exogenous sensory events entails rapid inhibition of the saccadic eye movement system, but the pathways mediating the inhibition are unknown. We devised novel behavioral paradigms in primates, involving multisensory stimulation, which allow neurophysiological experiments to isolate the contributions of individual sensory, motor, and sensory-motor brain areas to saccadic inhibition. Such paradigms underscore that inhibition involves distinct temporal and spatial processes that are dominated by the visual modality.

neuroscience↗

bioRxiv2025

Role of tankyrase scaffolding in the β-catenin destruction complex and WNT signaling

Aberrant WNT/{beta}-catenin signaling drives tumorigenesis and metastasis in cancer. Although enzymatic inhibitors of tankyrase (TNKS) effectively block AXIN degradation and stabilize the {beta}-catenin destruction complex (DC), they have demonstrated limited efficacy in various cancer models. Here we demonstrate that, unexpectedly, the induction of AXIN puncta represents a major barrier to achieving therapeutic efficacy. Mechanistically, catalytic inhibition of TNKS prevents TNKS turnover and drives its accumulation in the DC, wherein the scaffolding function of TNKS induces AXIN puncta formation, rigidifies the DC, and impedes {beta}-catenin turnover. Chemically induced degradation of TNKS overcomes this limitation by stabilizing AXIN without puncta formation, providing a deeper suppression of the WNT/{beta}-catenin pathway activity and the proliferation of colorectal cancer cells harboring dysfunctional APC mutations. Collectively, these findings provide an explanation for the unsatisfactory outcomes of drugging the WNT/{beta}-catenin signaling pathway by TNKS inhibitors and highlight TNKS degradation as a promising approach to treat WNT/{beta}-catenin-driven cancers.

cancer biology↗

bioRxiv2025

Convergent utilization of APN receptor by hedgehog merbecoviruses

Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for EriCoVs. Analysis of 94 APN orthologs indicates that EriCoVs have a limited host range, primarily utilizing hedgehog APN and, to a lesser extent, APN from certain felids, shaped by specific determinants at the virus-receptor interface. Cryo-EM reveals an APN-binding mode distinct from those used by alpha- and deltacoronaviruses. Functional assays indicate that hedgehog transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes pseudovirus entry. Neutralizing antibodies targeting RBD and APN were developed and could effectively block EriCoV pseudovirus entry and propagation. These findings reveal an unexpected convergent evolution of APN utilization among merbecovirus, establishing a foundation for risk assessment and countermeasure development. Graphic abstractConvergent utilization of hedgehog APN by EriCoVs O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/676546v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1ea334aorg.highwire.dtl.DTLVardef@cd53bforg.highwire.dtl.DTLVardef@352102org.highwire.dtl.DTLVardef@a1eed_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Reducing Foetal Bovine Serum Culture Conditions does not affect GPCR Signalling

Animal use in research extends beyond the use of animal models to study physiology and disease. Many aspects of in vitro research use reagents derived from animals, most prolifically the use of foetal bovine serum (FBS) in growth media for cellular models. With the aim to reduce animal use, we investigated the effect of reduced FBS culture conditions on cell growth, as well as different stages of G protein-coupled receptor (GPCR) signalling, a wide area of research which might therefore impact many groups. We identified little differences on cell growth or GPCR signalling when reducing culture FBS percentage from 10% to 5%, using assays ranging from receptor activation to downstream transcription factors stimulation. In addition to diminishing animal use, the reduction of FBS use will also have economic and environmental benefits, which we hope will be of benefit to the wider research community.

pharmacology and toxicology↗

bioRxiv2025

A ratio-based framework using Quartet reference materials for integrating long- and short-read RNA-seq

Long-read RNA sequencing (lrRNA-seq) enables full-length transcript profiling but is confounded by technical batch effects that compromise quantification and prevent data integration across platforms, protocols, and laboratories. The lack of a transcriptome-wide biological ground truth has hindered objective benchmarking. To address these dual challenges, we leveraged certified Quartet reference materials to generate one of the largest multi-center lrRNA-seq resources to date: over one billion long reads from 144 libraries across four PacBio and Nanopore protocols in four independent laboratories. We first establish that ratio-based quantification against built-in reference samples effectively removes technical noise, revealing underlying biological signals. We then constructed the first ratio-based reference datasets for full-length transcripts-- comprising 10,218 isoforms and 6,032 alternative splicing (AS) events--and orthogonally validated them with RT-qPCR. Finally, a comprehensive benchmark using these ground truths reveals that a hybrid strategy integrating long- and short-read data (hybrid-seq) achieves the highest quantification accuracy for both isoforms and AS events. Our work provides a foundational framework and resource for evaluating lrRNA-seq technologies and accelerating the standardization of full-length transcriptomics for research and clinical applications.

genomics↗

bioRxiv2025

Dual-LLM Adversarial Framework for Information Extraction from Research Literature

Information Extraction (IE) is a fundamental task in Natural Language Processing (NLP) that aims to automatically identify relevant information from unstructured or semi-structured data. Information extraction from lengthy research literature, particularly in multi-omics studies, faces significant challenges due to their complex narratives and extensive context. To address this, we present a novel dual-LLM adversarial framework in which one large language model (LLM) performs the extraction and another provides iterative feedback to refine the results. This process systematically reduces errors, enhances consistency across heterogeneous data sources, and converges toward more accurate outputs. We evaluated our approach against manual and single-LLM extraction, using LLMs as evaluators. Experimental results show that our adversarial framework outperforms these baselines, highlighting its effectiveness for extracting structured information from lengthy scientific texts.

bioinformatics↗

bioRxiv2025

Respiratory exposure to inert particulate matter during pregnancy leads to oxidative stress and cardiac proteome remodeling

Withdrawal StatementThe authors have withdrawn this manuscript because upon peer review major revisions have been identified as needed to ensure that the conclusions are in par with the presented data. Current studies are ongoing to provide a more complete version of the manuscript for peer review publication. Therefore, the authors do not wish this work to be cited as reference for the project in its current form. If you have any questions, please contact the corresponding author.

molecular biology↗

bioRxiv2025

wspA mutation mediated Pseudomonas aeruginosa rugose small colony variant and its hyper-biofilm

Pseudomonas aeruginosa is a major multidrug-resistant pathogen whose biofilm formation complicates treatment. P. aeruginosa rugose small colony variants (RSCVs), characterized by enhanced biofilm production and resistance, pose significant clinical challenges. However, the formation mechanisms remain unclear in clinical strains. Paired clinical P. aeruginosa isolates AR8023-1 (wild-type) and AR8023-2 (RSCV) were collected from urine samples of a neurosurgery inpatient chronologically. The genome comparison was performed using Breseq, and plasmid complementation was performed to construct complemented strain (AR8023-2RifR::wspAAR8023-1). Then, all the aforementioned strains were examined RSCV phenotype, biofilm formation, and motility, and gene expression differences induced by the wspA mutation were analyzed via transcriptome sequencing, followed by quantification of c-di-GMP. In addition, we assessed antimicrobial susceptibility of the strains under both planktonic and biofilm conditions. The results showed that both AR8023-1 and AR8023-2 were identified as ST3420. AR8023-1 exhibited typical smooth morphology, while AR8023-2 displayed a RSCV phenotype. A single SNP difference was identified between the two strains, characterized by a deletion of glutamine at position 289 (CAG triplet deletion) in the wspA gene of strain AR8023-2. The complement isolates restored wild-type morphology, motility, and biofilm formation. Transcriptomics revealed a significant upregulation of c-di-GMP metabolic genes in RSCVs (P < 0.001), indicating that the WspA{Delta}289Q mutation activates diguanylate cyclase (DGC) activity, thereby elevating c-di-GMP synthesis. Intracellular c-di-GMP levels were significantly higher in RSCVs than those in WT and complement isolates, respectively (P < 0.001). Consequently, biofilm susceptibility testing demonstrated the {beta}-lactams MBIC of RSCVs was [≥]8-512-fold higher than those of WT and complement isolates. This study identifies a clinical WspA{Delta}289Q mutation that elevates c-di-GMP, driving RSCV formation and biofilm-mediated resistance. IMPORTANCEP. aeruginosa is a leading multidrug-resistant pathogen whose biofilm formation capability greatly complicates treatment outcomes. This study identifies a novel clinical mutation in the wspA gene (WspA{Delta}289Q) that drives the emergence of rugose small colony variants (RSCVs), a phenotype linked to heightened biofilm production and antimicrobial resistance. We demonstrate that this mutation upregulates cyclic di-GMP synthesis, leading to increased biofilm formation and markedly reduced susceptibility to {beta}-lactam antibiotics under biofilm conditions. These results provide important insights into the genetic basis of RSCV development in clinical settings and highlight the role of c-di-GMP signaling in biofilm-associated resistance. Our findings underscore the need to target c-di-GMP pathways as a potential strategy for combating persistent P. aeruginosa infections.

microbiology↗

bioRxiv2025

SARS-CoV-2 nucleocapsid induces hyperinflammation and vascular leakage through the Toll-like receptor signaling axis in macrophages

Tens of thousands of severe COVID-19 cases are hospitalized weekly in the U.S., often driven by an imbalance between antiviral responses and inflammatory signaling, leading to uncontrolled cytokine secretion. The SARS-CoV-2 nucleocapsid (N) protein is a known immune antagonist, but its role in macrophage-driven cytokine storms is unclear. We demonstrate that N functions in a pathway-specific manner, specifically amplifying nuclear factor {kappa}B-related transcripts upon Toll-like receptor 7/8 stimulation. Moreover, we show that this is a conserved feature of pathogenic coronaviruses, with the delta variant N being the most pro-inflammatory. Our interaction networks suggest the delta variant N drives inflammation through interactions with several stress granule-related proteins. Profiling of secreted cytokines revealed that supernatants from the delta variant N-expressing macrophages disrupt brain and heart endothelial barriers, implicating N in COVID-19-associated cognitive and cardiac complications. Our findings highlight N-mediated immune imbalance as a driver of severe COVID-19 and identify N as a promising therapeutic target to mitigate hyperinflammation.

microbiology↗

bioRxiv2025

Core gastric microbiota linked to pathogenesis and preserved across age stratified cohorts

Mounting evidences reveal a complex microbial niche in gastric environment. Nevertheless, the phylogenetic stability of gastric microbiota remains undetermined. Here, using full-length 16S rRNA and ITS amplicon sequencing applied to gastric brushes from a pediatric endoscopic cohort (n=243), we identified a core microbiota comprising 11 genera, which clustered into two antagonistic communities (CST1A and CST1B). CST1A was enriched in healthy subjects, while CST1B correlated with gastroduodenal disease. Notably, this bipartite architecture arose through bacterial interactions, with fungal taxa demonstrating minimal integration. Further stratified analyses revealed that H. pylori infection preferentially depleted CST1A taxa. The subclusters exhibited divergent metabolic profiles, with CST1A specializing in motility processes and CST1B in nucleotide/peptidoglycan synthesis. Cross-cohort validation of six datasets (n=1,183) confirmed the persistent relevance of the CST1A-CST1B structure across disease stages. Collectively, our findings define a core gastric microbiota established in early life, linked to gastric disease risk, and persisting into adulthood. HighlightsO_LIIdentification of a gastric bacterial network established in early life. C_LIO_LIDysbiosis in two subclusters within the core microbiota is associated with pediatric peptic ulcers. C_LIO_LIHelicobacter pylori infection reduces the abundance of the core microbiota, with a particularly greater inhibitory effect on CST1A. C_LIO_LIThe core microbiota is preserved across age stratified cohorts and is associated with gastric cancer. C_LI

microbiology↗

bioRxiv2025

Human single-cell atlas analysis reveals heterogeneous endothelial signaling

Endothelial cells (ECs) play complex roles across tissues and vessel types. Yet, systematic investigations of EC heterogeneity in the combined context of vessel type and tissue microenvironment are still largely lacking. We integrated over three million single cells of scRNA-seq datasets in 15 human tissues and found that ECs in some tissues (e.g., heart and kidney) exhibited greater tissue specificity, while others displayed more substantial vessel specificity. We developed a computational pipeline to analyze cell-cell communications (CCC) mediated by metabolites or proteins to explore microenvironmental regulation. Interestingly, our results showed that CCC events involving ECs varied vastly across tissues, highlighting tissue-specific EC interactions. Using topic modeling, we identified CCC patterns, termed CCC topics, representing metabolite- and protein-mediated interactions between ECs and other tissue-resident cells. Most CCC topics exhibited high tissue specificity, potentially explaining microenvironmental regulations for EC heterogeneity. The work systematically investigates EC heterogeneity and provides insights into how EC heterogeneity was regulated across diverse tissue microenvironments.

bioinformatics↗

bioRxiv2025

Temporal control of sgRNA library activation unlocks large-scale in vivo CRISPR screens

Functional genomics screens have illuminated genetic dependencies in cancer, but conventional in vitro approaches fail to capture vulnerabilities shaped by the tumor microenvironment. Here, we implement CRISPR-StAR (Stochastic Activation by Recombination), a next-generation inducible CRISPR screening platform for large-scale in vivo applications. The system uses a dual lox-based recombination system to enable guide-level normalization and clonal knockout phenotyping. To analyze the rich (barcode-embedded) sequencing output, we developed UMIBB, a superior Bayesian statistical framework for quantifying gene-level dropout and enrichment compared to conventional software packages. Screening a 30,000-sgRNA library in A549 xenografts, followed by clone representation and dropout correlation analyses, showed high fidelity and reproducibility with dropout phenotypes resolvable using as few as 30 tumors for this size library. Validation across multiple tumor models demonstrated that a single tumor can provide reliable, functional annotation for [~]1,000 genes leveraging intra-tumor library controls for normalization. Comparing in vivo and in vitro screens revealed that a substantial subset of tumor suppressor genes exerts strong phenotypic effects only observable in vivo. For example, single-gene knockout and transcriptomic profiling confirmed that KMT2C and KMT2D have contrasting impacts on tumor growth - an insight that would have been overlooked in standard cell culture. Looking ahead, CRISPR-StAR screening, combined with our user-friendly analysis pipeline available on GitHub (R-package), offer an integrated framework for creating in vivo dependency maps that can complement existing vitro datasets like DepMap and Achilles. Critically, our approach reduces animal use by up to 7-fold compared to conventional in vivo dropout screens. This represents a significant ethical and methodological advancement - achieving genome-scale resolution with far fewer animals and greater reproducibility.

genomics↗