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Accurate quantification of CRISPR effects in pooled FACS screens using Waterbear

ABSTRACT: CRISPR screens are powerful tools to identify key genes that underlie biological processes. One important type of screen uses fluorescence activated cell sorting (FACS) to sort perturbed cells into bins based on the expression level of a marker gene, followed by guide RNA (gRNA) sequencing. Analysis of these data presents multiple statistical challenges due to multiple factors including the discrete nature of the bins and typically small numbers of replicate experiments. To address these challenges, we developed a robust and powerful Bayesian random effects model and software package called Waterbear. Furthermore, we used Waterbear to explore how various experimental design parameters affect statistical power to establish principled guidelines for future screens. Finally, we experimentally validated our model findings that, when using Waterbear for analysis, high power is maintained even at low coverage and a high multiplicity of infection. We anticipate that Waterbear will be of broad utility for analyzing FACS-based CRISPR screens.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE242880 | GEO | 2025/10/01

REPOSITORIES: GEO

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Project description: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.

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Accurate quantification of CRISPR effects in pooled FACS screens using Waterbear

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