CRISPR/Cas9 Transcription Factor Knockout Screen for Regulators of In Vitro Cardiac Differentiation

Utilizing a high-throughput functional genomic screen, we identified transcription factors that were functionally relevant in 2D and 3D stem cell-based cardiomyocyte (CM) differentiation systems by knocking out >1,600 TFs. We performed differentiations into 2D or 3D CM systems using a doxycycline-inducible Cas9 H9 stem cell line transduced with a comprehensive TF library. Once differentiated, CMs and non-CMs were sorted out based on TNNT2 expression and sequenced using NGS. Mageck analysis was performed to determine the guides enriched in non-CMs or CMs.