DCas9-mediated dysregulation of gene expression in human induced pluripotent stem cells during primitive streak differentiation

Abstract

CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.