Endogenous tagging of the TBXT (Brachyury) gene with 2A-EGFP in BJNhem19 human embryonic stem cells using CRISPR-Cas9 genome editing.
Brachyury (TBXT) is a T-box transcription factor essential for directing human embryonic stem cells (hESCs) toward mesodermal lineage specification. Here, we report the generation of a Brachyury/TBXT-2A-EGFP knock-in reporter line in the BJNhem19 hESC line using CRISPR/Cas9-mediated homology-directed repair. Accurate integration of the reporter cassette was confirmed by DNA sequencing and validated by the comparison of EGFP expression to endogenous TBXT gene expression following directed mesodermal differentiation. This reporter line provides a tractable fluorescence-based platform for real-time monitoring of mesodermal commitment, which will help in high-content screening of differentiation protocols.
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