Biology subjects

Yang, Y.

Publications and source records attributed to Yang, Y..

At least 487 records · Page 27Linked to original sources

bioRxiv2025

Highly potent novel multi-armoured IL13Rα2 CAR-T subverts the immunosuppressive microenvironment of Glioblastoma

Glioblastoma remains one of the most challenging and lethal brain cancers, with limited treatment options. While CAR-T cells have shown promise in some patients, sustaining T-cell activity and overcoming the immunosuppressive tumour microenvironment remain significant hurdles. Here, we present an armoured CAR-T cell design to address these challenges and enhance persistence in GBM tumours. We developed a highly specific humanised single-domain antibody targeting IL13R2 and included it alongside four additional modular elements in a single retroviral vector for CAR-T generation. Our results demonstrate that this single-cassette CAR-T cell design possesses high resilience against TGF-{beta}-mediated immunosuppression, enhanced tumour-killing capacity through IL-12 secretion while maintaining a favourable safety profile, extended persistence in the host, and an additional layer of safety control through the incorporation of a suicide switch. Importantly, despite its complexity, the construct can still be manufactured efficiently. These advancements represent a significant step forward in addressing key challenges associated with CAR-T cell therapy in solid tumours.

cancer biology↗

bioRxiv2025

Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

The liver lymphatic system plays a critical role in maintaining interstitial fluid balance and immune regulation. Efficient lymphatic drainage is essential for liver homeostasis, but its role in liver disease progression remains poorly understood. In cirrhosis, lymphangiogenesis initially compensates for increased lymph production, but impaired lymphatic drainage in advanced stages may lead to complications such as ascites and portal hypertension. This study aimed to evaluate how liver lymphatic dysfunction affects disease progression and to assess therapeutic strategies. Using a surgical model to block liver lymphatic outflow, we found that impaired drainage accelerates liver injury, fibrosis, and immune cell infiltration, even in healthy livers. Mechanistically, enhanced TGF-{beta} signaling in liver lymphatic endothelial cells (LyECs) contributed to reduced lymphatic vessel (LV) density and function in late-stage decompensated cirrhosis. This dysfunction was linked to the progression from compensated to decompensated cirrhosis, particularly in patients with primary sclerosing cholangitis (PSC). Conversely, liver-specific overexpression of VEGF-C via AAV8 improved lymphatic drainage, restored LV density, reduced fibrosis, mitigated liver injury, and alleviated portal hypertension in cirrhotic rats. These findings establish impaired liver lymphatic function as a pivotal driver of cirrhosis progression and identify VEGF-C as a promising therapeutic target to prevent decompensation.

pathology↗

bioRxiv2025

Origin of subgenomes in the circumboreal allopolyploid carnivorous plant Drosera anglica (Droseraceae)

Premise of StudyThe parentage of a widespread member of the carnivorous sundew genus Drosera, the allopolyploid Drosera anglica, remains uncertain despite over 100 years of morphological, cytological, and, more recently, molecular study. MethodsUsing transcriptomic and genomic data from 12 species Drosera sect. Drosera including four D. anglica populations and a disjunct Idaho population of D. intermedia, we assembled genes in HybPiper and phased sequences in HybPhaser. We estimated heterozygosity and generated flow cytometry data to assess ploidy levels. We estimated species relationships with phylogenetic and pairwise genetic distance methods. Additionally, we assembled rbcL and ITS reads to compare to previous data. Key ResultsSequences from phased subgenomes highly supported D. anglica as sister to D. rotundifolia and D. linearis, differing from previous analyses based on chromosome pairing and Sanger sequencing with limited taxon sampling. Both ITS and rbcL sequences of D. anglica were the most similar to D. linearis. Drosera anglica is intermediate between both parents in leaf shape and microhabitat; however, across D. sect. Drosera, neither leaf shape nor biogeographic distribution were reliable indicators of phylogenetic relationships. Despite a range-wide sampling, we did not find evidence for multiple origins of D. anglica. Additionally, we confirmed that the Idaho population previously identified as D. intermedia is D. anglica. ConclusionsDrosera anglica arose from allopolyploidy between D. linearis (the chloroplast donor) and D. rotundifolia. Our study demonstrates the importance of taxon sampling and careful examining complex phylogenomic data, and presents an exemplar of analyzing allopolyploid relationships in plant lineages.

evolutionary biology↗

bioRxiv2025

Collagen Hydrogel Tube Microbioreactors for Cell and Tissue Manufacturing

The production of mammalian cells in large quantities is essential for various applications. However, scaling up cell culture using existing bioreactors poses significant technical challenges and high costs. To address this, we previously developed an innovative 3D culture system, known as the AlgTube cell culture system, for high-density cell cultivation. This system involves processing cells into microscale alginate hydrogel tubes, which are suspended in the culture medium within a vessel. These hydrogel tubes shield cells from hydrodynamic stress and maintain the cell mass below 400 {micro}m in diameter, facilitating efficient mass transport and creating a favorable microenvironment for cell growth. Under optimized conditions, AlgTubes supported long-term culture with high cell viability, rapid expansion (1000-fold increase over 9 days per passage), and high yield (5x10 cells/mL), offering significant advantages over conventional methods. Despite these benefits, AlgTubes have critical drawbacks. They are mechanically fragile, with frequent breakage during culture leading to cell leakage and production failures. Additionally, many cell types exhibit poor growth due to the inability to adhere to the alginate surface, making alginate hydrogel microtubes unsuitable for industrial-scale cell production. To overcome these challenges, we developed a novel collagen hydrogel tube-based microbioreactor system in this work. This system provides enhanced robustness and adhesion, enabling scalable, cost-effective, and efficient cell production for a wide range of applications.

bioengineering↗

bioRxiv2025

Micromorphology and ammonium transmembrane transport characteristics in roots of rice seedlings

During the seedling stage, rice root exhibit rapid growth and absorb significant quantities of nutrients. This study involved cultivating rice seedlings in two different seedling-raising mediums characterized by high ammonium concentration (HN, NH4+ 1.2 mg g-1) and low ammonium concentration (LN, NH4+ 0.006 mg g-1). The morphology of distinct root zones (root cap, meristematic zone, elongation zone, and maturation zone) was observed under a microscope throughout root development, and variations in transmembrane flux rates of NH4+ in different root zones were assessed using Non-invasive micro-test technology (NMT). Results showed that the root caps of both seminal and adventitious roots became separated from the root tip between 9 and 12 days after root emergence. During this time, the root tip transformed from a sharp to a round shape, and the distance from the maturation zone to the root tip progressively decreased from over 900 m on 6 days after sowing (DAS) to under 500 m on DAS12. The meristematic zone was the primary site for NH4+ absorption in new root and proved to be particularly sensitive to environmental NH4+ concentration. At DAS 6, the net NH4+ flux rate was highest in the meristematic zone under both HN and LN treatments. Additionally, the external NH4+ concentration influenced the direction of NH4+ flux in the meristematic zone at DAS9, with HN being a net NH4+ influx and LN being an NH4+ efflux, and the net NH4+ influx in the meristematic zone persisted for 3 days longer in the HN treatment compared to the LN treatment. In mature roots, the root hair zone emerged as the primary site of NH4+ uptake, exhibiting an infNH4+ influx rate of 40-140 pmol cm-1 s-1, and this low rate of uptake could be sustained for up to 12 days after root emergence. By DAS15, a net NH4+ efflux was observed in the entire seminal root, signaling the loss of NH4+ absorption function 15 days post-emergence. A similar trend was noted in adventitious roots, where NH4+ uptake remained functional for 12-15 days after root emergence. As rice seedlings continued to grow, new adventitious roots replaced old ones, facilitating ongoing NH4+ uptake.

plant biology↗

bioRxiv2025

Optimization of Extraction Process by Response Surface Methodology of Total Flavonoids from Ziziphus Jujube Flesh and its Sedative-Hypnotic Effects

Background & aimsThis study optimizes the extraction process of total flavonoids from Ziziphus jujube flesh (TFZJF) by response surface methodology and investigating its sedative-hypnotic effects and mechanisms in a para-chlorophenylalanine (PCPA)-induced insomnia mouse model. It provides theoretical support for the further comprehensive utilization of Ziziphus jujube fruit and flesh. MethodsSingle-factor experiment and Box-Behnken response surface design were used to study the ethanol reflux extraction process of TFZJF, with flavonoid extraction rate as the indicator, to obtain the optimal extraction process of TFZJF. An insomnia model in mice was induced via an intraperitoneal injection of PCPA, and the effects of TFZJF on this model, along with its underlying mechanisms, were assessed using various approaches, including sodium pentobarbital-induced sleep potentiation, HE staining of tissue sections, ELISA, RT-PCR, WB, and serum metabolomics. ResultsThe results showed that the optimized extraction conditions for TFZJF included a solid-liquid ratio (SLR) of 1:25 g{middle dot}mL-1, ethanol concentration of 60%, and extraction time of 60 min, yielding an extraction efficiency of 1.98%. Data from the experimental groups indicated dose-dependent sleep improvement in the insomnia model, with the high-dose TFZJF (TFZJF-H) group exhibiting the most significant effect, followed by the medium-dose (TFZJF-M) and low-dose (TFZJF-L) groups. Metabolomic analysis revealed that TFZJF administration positively impacted the metabolic profile of PCPA-induced insomnia, particularly affecting pathways related to phenylalanine, tyrosine, cytochrome P450, and alanine metabolism on non-targeted metabolomics. ConclusionsThe extraction process is stable and reliable, which can be used for the extraction of TFZJF; meanwhile, TFZJF demonstrated significant sleep-enhancing effects in the PCPA-induced insomnia mouse model, supporting its potential for resource development and the utilization of non-medicinal parts of Ziziphus jujube.

biochemistry↗

bioRxiv2025

The RING Finger E3 Ligase RNF25 Protects DNA Replication Forks Independently of its Canonical Roles in Ubiquitin Signaling

The DNA damage response (DDR) mechanisms that allow cells to tolerate DNA replication stress are critically important for genome stability and cell viability. Using an unbiased genetic screen we identify a role for the RING finger E3 ubiquitin ligase RNF25 in promoting DNA replication stress tolerance. In response to DNA replication stress, RNF25-deficient cells generate aberrantly high levels of single-stranded DNA (ssDNA), accumulate in S-phase and show reduced mitotic entry. Using single-molecule DNA fiber analysis, we show that RNF25 protects reversed DNA replication forks generated by the fork remodeler HLTF from nucleolytic degradation by MRE11 and CtIP. Mechanistically, RNF25 interacts with the replication fork protection factor REV7 and recruits REV7 to nascent DNA after replication stress. The role of RNF25 in protecting replication forks is fully separable from its canonical functions in ubiquitin conjugation. This work reveals the RNF25-REV7 signaling axis as an important protective mechanism in cells experiencing replication stress.

molecular biology↗

bioRxiv2025

Improved Identification of Host Cell Proteins in Monoclonal Antibodies by Combining Filter Aided Sample Preparation and Native Digestion

Host cell proteins (HCPs) in biotherapeutics can present potential safety risks or compromise product stability at trace levels. Therefore, removal, testing, and characterization of HCPs are critical throughout biotherapeutic process development. While enzyme-linked immunosorbent assay (ELISA) is the gold standard for quantifying HCPs, it does not provide information on HCP identity. As a result, HCP analysis by liquid chromatography-mass spectrometry (LC-MS) has gained prominence for its ability to identifying HCPs. However, compared to ELISAs parts-per-billion sensitivity, LC-MS is limited by its dynamic range, often unable to cover magnitude difference between HCP and biotherapeutic concentrations. Thus, an effective HCP enrichment method is highly desirable. In this study, we propose a new strategy for HCP identification that combines filter-aided sample preparation (FASP) with native digestion, followed by shotgun proteomics analysis. This approach improves mAb removal compared to standard native digestion, enabling greater HCP enrichment and identification. Our method detects all spiked proteins at 1 ppm and most at 0.5 ppm, ranging from 12 to 470 kDa, demonstrating broad molecular weight coverage of HCPs. A proof-of-concept analysis using the NISTmAb standard demonstrates that our filter-aided native digestion identifies 155 more HCPs than the standard native digestion. When applying this strategy to an in-house antibody with low amounts of HCPs, we can quantify HCPs at levels as low as 0.03 ppm, demonstrating the high sensitivity in HCP characterization.

biochemistry↗

bioRxiv2025

Role of RSPO3 in Estrogen-mediated Sex Differences in Body Fat Distribution: A Single-cell Data-driven Study

Obesity is a global health concern affecting more than one-third of the adult population worldwide. Epidemiological studies indicate that body fat distribution, more than overall adiposity, is a key risk factor for obesity-related diseases. Genome-wide association studies revealed that RSPO3 is associated with body fat distribution. Additionally, marked sex differences in body fat distribution are largely influenced by estrogen, suggesting that exploring the relationship between estrogen and RSPO3 expression may illuminate the mechanisms underlying the sexual dimorphism of fat distribution. We revisited a comprehensive set of gene expression data from humans and mice to explore these mechanisms. We assessed RSPO3 expression in male and female adipose tissues and utilized single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics to examine RSPO3s function within different tissues. Our findings confirmed significantly higher RSPO3 expression in male adipose tissue compared to female. Furthermore, our data for the first time suggest that estrogen plays a crucial role in fat distribution, as blocking estrogen signaling upregulates RSPO3 expression. In mouse liver, we observed a correlation between sex-specific ADK overexpression and RSPO3 expression, resulting in increased obesity and hepatocyte expansion via WNT/{beta}-catenin signaling in male mice. This study sheds light on how estrogen influences sexual dimorphism in fat distribution and underscores RSPO3s vital role in this process. These insights open new avenues for targeted therapeutic interventions addressing obesity and metabolic diseases with a focus on sex-specific factors.

bioinformatics↗

bioRxiv2025

Pathobiont-triggered induction of epithelial IDO1 drives regional susceptibility to Inflammatory Bowel Disease

The structure and function of the mammalian gut vary by region, yet why inflammatory diseases manifest in specific regions and not others remains unclear. We use a TNF-overexpressing Crohns disease (CD) model (Tnf{Delta}ARE/+), which typically presents in the terminal ileum (TI), to investigate how environmental factors interact with the hosts immune susceptibility to drive region-specific disease. We identified Chlamydia muridarum, an intracellular bacterium and murine counterpart to the human sexually transmitted C. trachomatis, as necessary and sufficient to trigger disease manifestation in the ascending colon (AC), another common site of human CD. Disease manifestation in the AC depends on indoleamine 2,3-dioxygenase (IDO1) activity induced by hypersensitive surface secretory cells in genetically susceptible hosts. Single-cell and microbial analyses of human specimens also implicates this pathobiont-epithelial IDO1 pathway in patients with a history of CD in the AC. Our findings demonstrate that genetic and microbial factors can independently drive region-specific disease and provide a unique model to study CD specific to the AC.

cell biology↗

bioRxiv2025

Roles of spoVF operon subunits A and B in regulating cell morphology and biofilm formation in the biocontrol agent Bacillus subtilis

Enhanced expression of spoVF operon (sporulation initiation factor; dipicolinic acid (DPA) synthase) in Bacillus subtilis vegetative cells has been reported to increase host attachment and biocontrol ability. However, how the spoVF operon regulates these essential biocontrol factors remains unknown. In this study, it was found that the knockout of the spoVFA or spoVFB subunits, individually ({Delta}A and{Delta} B), or together ({Delta}A{Delta}B), decreased DPA biosynthesis, host attachment, nutrient competition ability, and biocontrol efficacy in B. subtilis. Moreover, the cell shape of the knockout strains changed from short rod to spherical. qRT-PCR analysis indicated that the spoVF operon regulates TCA cycle expression and biofilm formation by balancing the amounts of various transcriptional factors, such as spo0A, SinR, and AbrB, and the cofactor NADPH. The regulatory mechanism of the spoVF operon in B. subtilis biofilm-related genes was proposed for the first time. IMPORTANCEBacillus subtilis SpoVF operon contains the spoVFA or spoVFB subunits, which are responsible for the dipicolinic acid synthase activity. Dipicolinic acid is an important component of B. subtilis spores. In this study, the roles of spoVFA or spoVFB subunits in the biocontrol ability B. subtilis were examined, indicating that the spoVFA subunit plays a key role in B. subtilis biofilm formation, cell morphology, growth, and nutrient competition ability. Additionally, the regulatory network connecting the spoVF operon with biofilm formation-related genes of B. subtilis was proposed. The results obtained in this study help to understand key genes involved in the biocontrol ability of B. subtilis.

microbiology↗

bioRxiv2024

RNAGenesis: Foundation Model for Enhanced RNA Sequence Generation and Structural Insights

RNA molecules are central to gene regulation, catalysis, and molecular recognition, and offer broad opportunities for therapeutic applications. However, uncovering their complex sequence, structure, and function relationships, particularly for non-coding RNAs, remains a formidable challenge. Here, we introduce RNAGenesis, a Generalist RNA foundation model that integrates sequence representation, structural prediction, and de novo functional design within a single generative framework. Trained on diverse clustered non-coding RNAs, RNAGenesis leverages a BERT-style encoder, query-based latent compression, and a diffusion-guided decoder enhanced by inference-time alignment with gradient guidance and beam search strategies. Through comprehensive evaluations, RNAGenesis achieves state-of-the-art performance on 11 of 13 tasks in the BEACON benchmark and surpasses structure-aware models in inverse folding, 3D structure prediction, and de novo structure design. We further introduce RNATx-Bench, a dedicated benchmark for RNA therapeutics comprising over 100,000 experimentally validated sequences. RNAGenesis demonstrates strong predictive performance across ASOs, siRNAs, shRNAs, circRNAs, and untranslated region (UTR) variants. Furthermore, RNAGenesis enables functional RNA design, including aptamers targeting IGFBP3 and structurally constrained sgRNA scaffolds. Wet-lab validation confirms aptamer binding with KD values as low as 4.02 nM and up to 2.5-fold improvement in editing efficiency across CRISPR-Cas9, base editing, and prime editing systems. These results position RNAGenesis as a next-generation general-purpose RNA foundation model with broad utility for computational modeling and experimental therapeutic design.

bioinformatics↗

bioRxiv2024

In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors

Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.

neuroscience↗

bioRxiv2024

Spatiotemporal Regulatory Logics of Mouse Gastrulation

Spatiotemporal coordination of cellular and molecular events is crucial for cell fate commitment during mouse gastrulation. However, the high-precision mechanisms governing the timing and spatial dynamics remain poorly understood. Here, we present a time-series single-cell multi-omic dataset from the mouse gastrulating embryos and construct a hierarchical gene regulatory landscape. Integrating this with real three-dimensional transcriptomic coordinate, we created ST-MAGIC and ST-MAGIC (+) atlas, dissecting the spatiotemporal logics of regulatory networks and signaling responsiveness underpinning the lineage commitment at gastrulation. Specifically, we delineated the multi-omic basis for left-right symmetry breaking events in the gastrula and also revealed the spatiotemporal molecular relay for axial mesendoderm lineage, where early and intermediate transcription factors first open the chromatin regions and setup the responsiveness to signaling, followed by terminal factors to consolidate the transcriptomic architecture. In summary, our study presents a spatiotemporal regulatory logic framework of mouse gastrulation, that advances our understanding of mammalian embryogenesis.

developmental biology↗

bioRxiv2024

Combined inhibition of SHP2 overcomes adaptive resistance to type 1 BRAF inhibitors in BRAF V600E-driven high-grade glioma

BRAF-mutant gliomas can be therapeutically targeted with BRAF mutant-selective inhibitors, yet responses are often transient due to short-term adaptive or long-term treatment-emergent resistance. We hypothesized that vertical inhibition of multiple signaling nodes could improve the durability of BRAF inhibition and prevent or overcome adaptive resistance. Using human tissue samples, we identified frequent RAS pathway reactivation in gliomas resistant to BRAF inhibitors, suggesting a common escape mechanism. Using patient-derived cell lines, we observed that upregulation of RAS activity was an adaptive response to BRAFi and that knockdown of SHP2, a central regulator of RAS activity, resulted in enhanced sensitivity to BRAF or MEK inhibition. Moreover, combined small molecule inhibition with SHP2 and BRAF or MEK inhibitors increased the depth and durability of ERK pathway inhibition, as well as prevented paradoxical upregulation of RAS activity. RNA sequencing analysis revealed deeper suppression of ERK transcriptional output with combined therapy, along with decreased reactivation of EGFR. Combined SHP2/BRAF small molecule inhibitors prevented growth and induced cell death in some cell line models. In cell lines with treatment-emergent resistance, moreover, combined SHP2 and BRAF inhibition overcame resistance to BRAF inhibitor monotherapy. In vivo orthotopic and patient-derived xenograft models confirmed enhanced tumor growth inhibition with combined therapy. Together, our findings demonstrate the critical role of RAS/ERK signaling reactivation in driving resistance to BRAF inhibition in glioma, and demonstrate the potential utility for adding SHP2 inhibitors to overcome resistance in BRAF V600E mutant glioma. SignificanceThe addition of a SHP2i to BRAFi in BRAF-V600E glioma cells prevents tumor growth and can overcome resistance to BRAFi in preclinical models in vitro and in vivo.

cancer biology↗

bioRxiv2024

Recovery of highly contiguous genomes from complex terrestrial habitats reveals over 15,000 novel prokaryotic species and expands characterization of soil and sediment microbial communities

Genomes are fundamental to understanding microbial ecology and evolution. The emergence of high-throughput, long-read DNA sequencing has enabled recovery of microbial genomes from environmental samples at scale. However, expanding the microbial genome catalogue of soils and sediments has been challenging due to the enormous complexity of these environments. Here, we performed deep, long-read Nanopore sequencing of 154 soil and sediment samples collected across Denmark and through an optimised bioinformatics pipeline, we recovered genomes of 15,314 novel microbial species, including 4,757 high-quality genomes. The recovered microbial genomes span 1,086 novel genera and provide the first high-quality reference genomes for 612 previously known genera, expanding the phylogenetic diversity of the prokaryotic tree of life by 8 %. The long-read assemblies also enabled the recovery of thousands of complete rRNA operons, biosynthetic gene clusters and CRISPR-Cas systems, all of which were underrepresented and highly fragmented in previous terrestrial genome catalogues. Furthermore, the incorporation of the recovered MAGs into public genome databases significantly improved species-level classification rates for soil and sediment metagenomic datasets, thereby enhancing terrestrial microbiome characterization. With this study, we demonstrate that long-read sequencing and optimised bioinformatics, allows cost-effective recovery of high-quality microbial genomes from highly complex ecosystems, which remain the largest untapped source of biodiversity for expanding genome databases and filling in the gaps of the tree of life.

bioinformatics↗

bioRxiv2024

Near 100% genome editing efficiency with a one-step all RNA prime editing system in a human reporter iPSC line

CRISPR/Cas9 nucleases offer powerful tools for genome editing but can cause unintended mutations at the target site due to the creation of double-stranded DNA breaks. Prime editing (PE) is arguably a safer technology as it relies on the creation of single-strand DNA nicks and avoids possible indels. Despite its precision, current PE systems are limited by relatively low editing efficiency in non-immortalized cells. Here, we optimized prime editing in human induced pluripotent stem cells (hiPSCs) using a fluorescence-based assay to achieve near-complete editing of a BFP transgene. Using an all-RNA approach, precise prime editing of a single nucleotide variant was observed in >95% of cells with minimal unintended edits at the target site.

molecular biology↗

bioRxiv2024

Comparison and evaluation of methods to infer gene regulatory networks from multimodal single-cell data

Cells regulate their functions through gene expression, driven by a complex interplay of transcription factors and other regulatory mechanisms that together can be modeled as gene regulatory networks (GRNs). The emergence of single-cell multi-omics technologies has driven the development of several methods that integrate transcriptomics and chromatin accessibility data to infer GRNs. While these methods provide examples of their utility in discovering new regulatory interactions, a comprehensive benchmark evaluating their mechanistic and predictive properties as well as their ability to recover known interactions is lacking. To address this, we built a comprehensive framework, Gene Regulatory nETwork Analysis (GRETA), available as a Snakemake pipeline, that includes state of the art methods decomposing their different steps in a modular manner. With it, we found that the GRNs were highly sensitive to methods choices, such as changes in random seeds, or replacing steps in the inference pipelines, as well as whether they use paired or unpaired multimodal data. Although the obtained networks performed well in predictive evaluation tasks and partially recovered known interactions, they struggled to capture causal relationships from perturbation assays. Our work brings attention to the challenges of inferring GRNs from single-cell omics, offers guidelines, and presents a flexible framework for developing and testing new approaches. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/629764v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@11a452forg.highwire.dtl.DTLVardef@1b44cb9org.highwire.dtl.DTLVardef@190dbdorg.highwire.dtl.DTLVardef@d4f66b_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗