An approach for accelerated isolation of genetically manipulated cell clones with reduced clonal variability

Abstract

Genomic editing methods, such as the CRISPR/Cas9 system, are routinely used to study gene function in somatic cells. Due to the heterogeneity of mutations, it is necessary to purify cell clones grown in high dilution until colony formation, which can be a time consuming process. Here we tested a modified approach in which we seeded cells in high dilution, together with non-edited carrier cells. As a comparison, cells were also grown in high dilution with conditioned medium from a high density culture. When using carrier cells or conditioned medium, the formation of cell colonies is accelerated. Additionally, clones grown with carrier cells are more similar to the parental lines in terms of their tumorigenic properties. Surprisingly, key signaling cascades are very divergent between clones isolated from low density cultures, even with conditioned medium, in contrast to clones isolated with carrier cells. Thus, our study uncovers a significant limitation using the common approach of isolating cell clones following genetic modifications and suggests an alternative method that mitigates the problem of heterogeneity of gene expression between clones.\n\nSummary statementWe show here that standard methods of generating cell clones after CRISPR/Cas9 treatment result in highly divergent signaling cascades among clones. We demonstrate here an improved alternative approach.