Wild-type Heterogeneity Contributes to Clonal Variability in Genome-Edited Cells

Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype-phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts when comparing two populations of wild-type cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. Expression profiling of polyclonal vs. monoclonal mIMCD-3 wild-type cells and expression profiling of monoclonal vs. subclone mIMCD-3 cells. Data of polyclonal wild-type cells have been analyzed in a different context before (GSE179947).