Single Cell Transcriptional Perturbome in Pluripotent Stem Cell Models

Functional genomics screens in human induced pluripotent stem cell (hiPSC) models offer immense potential yet remain challenging. We developed iPS2-seq: iPS-optimized inducible Postranscriptional Silencing in pool deconvoluted by single cell sequencing. This method allows phenotype-agnostic screens in hPSCs and their derivatives through loss-of-function perturbations that are mRNA-depleting (bypassing genotoxicity), clonally traced (controlling genetic and epigenetic variability), single cell aware (accounting for asyncronous and heterogeneous differentiation), isogenic engineered (preventing silencing), inducible and reversible (enabling stage-specific studies and robust control-treatment paired analyses). iPS2-seq is compatible with both commercial microfluidics and homebrew split-pool scRNA-seq protocols, enabling screens with different scales, costs, and input materials. A dedicated bioinformatics pipeline, catcheR, supports iPS2-seq design and analysis. We demonstrate this by studying congenital heart disease-associated genes in hiPSC-derived monolayer cardiomyocytes and cardiac organoids, identifying a key role for SMAD2 in cardiomyogenesis. iPS2-seq strengthens and democratizes access to functional single cell genomics in and beyond hiPSC-derived organoids.