Motosugi Nami, Hasegawa Keita, Kurosaki Natsumi, Kawaguchi Erika, Izumi Kenji, Iida Yumi, Higashiseto Misaki, Yokoyama Keiko, Sasaki Ayumi, Nakabayashi Kazuhiko, Fukuda Atsushi
Department of Molecular Life Sciences, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Department of Maternal-Fetal Biology, National Center for Child Health and Development, Tokyo, Japan.
Stem Cell Res Ther. 2025 Jul 18;16(1):389. doi: 10.1186/s13287-025-04501-4.
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.
由于长链非编码RNA XIST的抑制导致X染色体失活（XCI）的不可逆侵蚀，这对疾病建模提出了重大挑战，并因X连锁基因的异常过表达而引发了对女性人类多能干细胞（hPSC）临床应用的安全担忧。虽然靶向XIST启动子的Cas9介导的非同源末端连接（NHEJ）可以诱导DNA去甲基化并通过重新激活XIST来恢复XCI，但其效率仍然很低。在这里，我们介绍了一种高效的策略，通过在Cas9介导的NHEJ过程中将TP53抑制与DNA甲基化维持抑制相结合来重新激活XIST。这种双重抑制方法将XIST阳性hPSC的比例从约5%提高到约43.7%，为在女性hPSC中稳定XCI以用于各种应用提供了一种强大的方法。
