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

• Dual inhibition of TP53 and DNA methylation maintenance during Cas9-mediated NHEJ significantly enhances XIST reactivation efficiency in female hPSCs, increasing from ~5% to ~43.7%. • This strategy provides a robust method for stabilizing X-chromosome inactivation, addressing a critical barrier in disease modeling and clinical applications of female hPSCs. • The approach avoids the use of exogenous sequences, mitigating concerns about unintended effects on XIST expression compared to HDR-based methods. • The findings have implications for improving the safety and reliability of female hPSCs in regenerative medicine and X-linked disorder research.