Generation of a LEUTX-2A-mCherry knock-in H1 human embryonic stem cell line using CRISPR/Cas9 system

Abstract

LEUTX is exclusively expressed at the 8-cell stage and serves as a key regulator of human embryonic genome activation. Induced 8-cell-like cells (8CLCs) derived from pluripotent stem cells offer a tractable model to dissect the molecular transition from pluripotency to totipotency. However, current 8CLC induction protocols are hampered by low efficiency and cellular heterogeneity. To overcome these limitations, we developed a fluorescent reporter system that dynamically monitors endogenous LEUTX expression, facilitating the purification and functional characterization of bona fide 8CLCs. This tool enables systematic interrogation of the regulatory networks underlying totipotency acquisition.