Biology subjects

De Jonghe, J.

Publications and source records attributed to De Jonghe, J..

5 recordsLinked to original sources

bioRxiv2026

Scalable variant-to-function screens using prime editing perturb-seq

Linking genetic variants to their functional consequences at scale remains a major challenge in human genetics. Perturb-seq is limited in the variants it can install, while prime editing has largely been restricted to simpler mechanistic readouts. Here, we introduce prime editing perturb-seq (PEP-seq), which links programmed mutations to transcriptomic states. We designed 2,164 prime editing guides targeting common expression quantitative trait loci (eQTLs), premature termination codons, and position-matched synonymous controls, generating 749,110 time-resolved single-cell transcriptomes. PEP-seq precisely detected nonsense-mediated decay, while probe-based junction measurements suggested depletion of mature RNAs. For eQTLs, a locus-size-corrected specificity metric showed that the intended target was frequently the most affected transcript within a 1 Mb window, with effects also associated with open chromatin near the edit site. These results establish PEP-seq as a scalable platform for direct, endogenous variant-to-function mapping.

genomics↗

bioRxiv2023

Saturation Genome Editing Resolves the Functional Spectrum of Pathogenic VHL Alleles

To maximize the impact of precision medicine approaches, it is critical to accurately identify genetic variants in cancer-associated genes with functional consequences. Yet, our knowledge of rare variants conferring clinically relevant phenotypes and the mechanisms through which they act remains highly limited. A tumor suppressor gene exemplifying the challenge of variant interpretation is VHL. VHL encodes an E3 ubiquitin ligase that regulates the cellular response to hypoxia. Germline pathogenic variants in VHL predispose patients to tumors including clear cell renal cell carcinoma (ccRCC) and pheochromocytoma, and somatic VHL mutations are frequently observed in sporadic renal cancer. Here, we optimize and apply Saturation Genome Editing (SGE) to assay nearly all possible single nucleotide variants (SNVs) across VHLs coding sequence. To delineate mechanisms, we quantify mRNA dosage effects over time and compare effects in isogenic cell lines. Function scores for 2,268 VHL SNVs identify a core set of pathogenic alleles driving ccRCC with perfect accuracy, inform differential risk across tumor types, and reveal novel mechanisms by which variants impact function. These results have immediate utility for classifying VHL variants encountered in both germline testing and tumor profiling and illustrate how precise functional measurements can resolve pleiotropic and dosage-dependent genotype-phenotype relationships across complete genes.

genomics↗

bioRxiv2022

Stem cell-derived mouse embryos develop within an extra-embryonic yolk sac to form anterior brain regions and a beating heart

Embryo-like structures generated from stem cells can achieve varying developmental milestones, but none have been shown to progress through gastrulation, neurulation, and organogenesis.1-7 Here, we show that "ETiX" mouse embryos, established from embryonic stem cells aggregated with trophoblast stem cells and inducible extraembryonic endoderm stem cells, can develop through gastrulation and beyond to undertake neural induction and generate the progenitors needed to create the entire organism. The head-folds of ETiX embryos show anterior expression of Otx2, defining forebrain and midbrain regions that resemble those of the natural mouse embryo. ETiX embryos also develop beating hearts, trunk structures comprising a neural tube and somites, tail buds containing neuromesodermal progenitors and primordial germ cells, and gut tubes derived from definitive endoderm. A fraction of ETiX embryos show neural tube abnormalities, which can be partially rescued by treatment with the metabolically active form of folic acid, reminiscent of common birth defects and therapies in humans. Notably, ETiX embryos also develop a yolk sac with blood islands. Overall, ETiX embryos uniquely recapitulate natural embryos, developing further than any other stem-cell derived model, through multiple post-implantation stages and within extra-embryonic membranes.

developmental biology↗

bioRxiv2022

Plakoglobin is a mechanoresponsive regulator of naïve pluripotency

Biomechanical cues are instrumental in guiding embryonic development and cell differentiation. Understanding how these physical stimuli translate into transcriptional programs could provide insight into mechanisms underlying mammalian pre-implantation development. Here, we explore this by exerting microenvironmental control over mouse embryonic stem cells (ESCs). Microfluidic encapsulation of ESCs in agarose microgels stabilized the naive pluripotency network and specifically induced expression of Plakoglobin (Jup), a vertebrate homologue of {beta}-catenin. Indeed, overexpression of Plakoglobin was sufficient to fully re-establish the naive pluripotency gene regulatory network under metastable pluripotency conditions, as confirmed by single-cell transcriptome profiling. Finally, we found that in the epiblast, Plakoglobin was exclusively expressed at the blastocyst stage in human and mouse embryos - further strengthening the link between Plakoglobin and naive pluripotency in vivo. Our work reveals Plakoglobin as a mechanosensitive regulator of naive pluripotency and provides a paradigm to interrogate the effects of volumetric confinement on cell-fate transitions. HighlightsO_LI3D agarose spheres stabilize the naive pluripotency network in mouse ESCs. C_LIO_LIVolumetric confinement induces expression of Plakoglobin, a vertebrate homologue of {beta}-catenin. C_LIO_LIPlakoglobin expression in the epiblast is specific to pre-implantation human and mouse embryos. C_LIO_LIPlakoglobin overexpression maintains naive pluripotency independently of {beta}-catenin. C_LI

developmental biology↗

bioRxiv2021

Droplet-based Single-cell Total RNA-seq Reveals Differential Non-Coding Expression and Splicing Patterns during Mouse Development

In recent years, single-cell transcriptome sequencing has revolutionized biology, allowing for the unbiased characterization of cellular subpopulations. However, most methods amplify the termini of polyadenylated transcripts capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts. Additionally, most workflows do not sequence the full transcript hindering the analysis of alternative splicing. We therefore developed VASA-seq to detect the total transcriptome in single cells. VASA-seq is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to over 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. The dynamics of the total single-cell transcriptome result in the discovery of novel cell type markers many based on non-coding RNA, an in vivo cell cycle analysis and an improved RNA velocity characterization. Moreover, it provides the first comprehensive analysis of alternative splicing during mammalian development.

molecular biology↗