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<p>The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA <i>XIST</i> 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 <i>XIST</i> promoter can induce DNA demethylation and restore XCI by reactivating <i>XIST</i>, its efficiency remains low. Here, we introduce a highly efficient strategy for <i>XIST</i> reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of <i>XIST</i>-positive hPSCs from ~ 5 to ~ 43.7%, providing a robust method for stabilizing XCI in female hPSCs for diverse applications.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月18日

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  • Nami Motosugi,
  • Keita Hasegawa,
  • Natsumi Kurosaki,
  • Erika Kawaguchi,
  • Kenji Izumi,
  • Yumi iida,
  • Misaki Higashiseto,
  • Keiko Yokoyama,
  • Ayumi Sasaki,
  • Kazuhiko Nakabayashi,
  • Atsushi Fukuda

摘要

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.