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

Stem Cell Res Ther. 2025 Jul 18;16(1):389. doi: 10.1186/s13287-025-04501-4.

Abstract

The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA XIST presents a major challenge for disease modeling and raises safety concerns for the clinical application of female human pluripotent stem cells (hPSCs) due to the aberrant overexpression of X-linked genes. While Cas9-mediated non-homologous end joining (NHEJ) targeting the XIST promoter can induce DNA demethylation and restore XCI by reactivating XIST, its efficiency remains low. Here, we introduce a highly efficient strategy for XIST reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of XIST-positive hPSCs from ~ 5 to ~ 43.7%, providing a robust method for stabilizing XCI in female hPSCs for diverse applications.

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • DNA Methylation* / genetics
  • Female
  • Gene Editing* / methods
  • Humans
  • Pluripotent Stem Cells* / cytology
  • Pluripotent Stem Cells* / metabolism
  • RNA, Long Noncoding* / genetics
  • RNA, Long Noncoding* / metabolism
  • Tumor Suppressor Protein p53* / antagonists & inhibitors
  • Tumor Suppressor Protein p53* / genetics
  • Tumor Suppressor Protein p53* / metabolism
  • X Chromosome Inactivation / genetics

Substances

  • RNA, Long Noncoding
  • XIST non-coding RNA
  • Tumor Suppressor Protein p53
  • TP53 protein, human