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

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 specific 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. In a proof-of-concept experiment, we show how monoclonal, genetically barcoded EBs enable us to better quantify the consequences of TF perturbations as well as “inter-individual” heterogeneity across EBs harboring the same genetic perturbation. Looking forward, monoclonal EBs and EB-derived organoids may be powerful tools not only for genetic screens, but also for modeling Mendelian disorders, as their underlying genetic lesions are overwhelmingly constitutional (i.e. present in all somatic cells), yet give rise to phenotypes with incomplete penetrance and variable expressivity. To explore the function of individual TFs in cell fate determination, a monoclonal CRISPRcut or CRISPRi mouse ES line was transduced at low multiplicity of infection with a lentiviral CROP-seq library of gRNAs targeting TFs and containing GFP and puromycin selection markers. After optional puromycin selection to titrate the fraction of perturbed cells, ES cells were self-aggregated into mosaic embryoid bodies (EBs). After 21 days, GFP-positive cells were subjected to scRNA-seq and gRNA enrichment PCR. In a subsequent experiment, we aimed to overcome the mosaic screen bottleneck by generating a pool of monoclonal EBs through sorting and seeding piggyFlex mESCs at low density on mouse embryonic fibroblasts. Individual mESCs grew into colonies for 5 days followed by lifting with Collagenase IV treatment and gentle agitation. Colonies, now clonal aggregates, were then differentiated into EBs on low adherent plates for 8 days. We further perturbed TFs in these monoclonal EBs and profiled them using scRNA-seq.