Barcoded monoclonal embryoids are a potential solution to confounding bottlenecks in mosaic organoid screens

作者：Samuel G. Regalado, Chengxiang Qiu, Jean‐Benoît Lalanne, Beth Martin, Madeleine Duran, Cole Trapnell, Aidan Keith, Silvia Domcke, Jay Shendure · 发表于：bioRxiv (Cold Spring Harbor Laboratory) · 年份：2025 · DOI：10.1101/2025.05.23.655669 · 被引用次数：4 · 研究领域：Pluripotent Stem Cells Research、Renal and related cancers、Reproductive Biology and Fertility

Abstract Genetic screens in organoids hold tremendous promise for accelerating discoveries at the intersection of genomics and developmental biology. Embryoid bodies (EBs) are self-organizing multicellular structures that recapitulate aspects of early mammalian embryogenesis. We set out to perform a CRISPR screen perturbing all transcription factors (TFs) in murine EBs. Specifically, a library of TF-targeting guide RNAs (gRNAs) was used to generate mouse embryonic stem cells (mESCs) bearing single TF knockouts. Aggregates of these mESCs were induced to form mouse EBs, such that each resulting EB was ’mosaic’ with respect to the TF perturbations represented among its constituent cells. Upon performing single cell RNA-seq (scRNA-seq) on cells derived from mosaic EBs, we found many TF perturbations exhibiting large and seemingly significant effects on the likelihood that individual cells would adopt certain fates, suggesting roles for these TFs in lineage specification. However, to our surprise, these results were not reproducible across biological replicates. Upon further investigation, we discovered cellular bottlenecks during EB differentiation that dramatically reduce clonal complexity, curtailing statistical power and confounding interpretation of mosaic screens. Towards addressing this challenge, we developed a scalable protocol in which each individual EB is monoclonally derived from a single mESC and genetically barcoded. In a proof-of-concept experiment, we show how the...