Highly efficient XIST reactivation in female hPSC by transient dual inhibition of TP53 and DNA methylation during Cas9 mediated genome editing

Abstract

Erosion of X-chromosome inactivation (XCI) complicates disease and developmental modeling in female human pluripotent stem cells (hPSCs). Previous studies demonstrated that Cas9-mediated editing of the XIST promoter via non-homologous end joining (NHEJ) or homology-directed repair (HDR) with a selection cassette upstream of XIST can trigger DNA demethylation and XIST reactivation, restoring XCI. Here, we show that NHEJ-mediated XCI reacquisition is more stable during differentiation than HDR. We further developed a novel, efficient XIST reactivation method by combining TP53 inhibition with DNA methylation maintenance suppression during Cas9-mediated NHEJ, offering a robust approach to achieving stable XCI in female hPSCs for diverse applications.