Female iPSC X chromosome inactivation erosion and its transcriptomic effects during CRISPR gene editing and neural differentiation.

Human induced pluripotent stem cells (hiPSCs), together with CRISPR editing, are commonly used for studying neurodevelopmental disorders. Female iPSCs gradually lose the expression of the noncoding X chromosome inactive-specific transcript (XIST), leading to random X chromosome inactivation (XCI) erosion that affects both X-linked and autosomal gene expression. However, the effects of CRISPR editing and neural differentiation on XCI erosion and how this may confound a real-world analysis of differentially expressed genes (DEGs) are poorly understood. Here, we analyzed hundreds of RNA sequencing (RNA-seq) samples of CRISPR-edited iPSC lines for 66 genes and the matched single-neuron RNA-seq data. We found variable XCI in CRISPR-edited female iPSCs, which was largely preserved in iPSC-derived neurons and predominantly influenced X-linked genes. Mechanistically, XIST mediates allele-specific epigenetic regulation of both X-linked and autosomal genes, with the former showing stronger allele-specific expression (ASE) bias that affects neurodevelopmental genes. These results have significant implications in hiPSC-based modeling of neurodevelopmental disorders.