CHD-associated enhancers shape human cardiomyocyte lineage commitment
Enhancers orchestrate gene expression programs that drive multicellular development and lineage commitment. Thus, genetic variants at enhancers are thought to contribute to developmental diseases by altering cell fate commitment. However, while many variant-containing enhancers have been identified, studies to endogenously test the impact of these enhancers on lineage commitment have been lacking. We perform a single-cell CRISPRi screen to assess the endogenous roles of 25 enhancers and putative cardiac target genes implicated in genetic studies of congenital heart defects (CHD). We identify 16 enhancers whose repression leads to deficient differentiation of human cardiomyocytes (CMs). A focused CRISPRi validation screen shows that repression of TBX5 enhancers delays the transcriptional switch from mid- to late-stage CM states. Endogenous genetic deletions of two TBX5 enhancers phenocopy epigenetic perturbations. Together, these results identify critical enhancers of cardiac development and suggest that misregulation of these enhancers could contribute to cardiac defects in human patients. HIGHLIGHTSO_LISingle-cell enhancer perturbation screens during human cardiomyocyte differentiation. C_LIO_LIPerturbation of CHD-linked enhancers/genes causes deficient CM differentiation. C_LIO_LIRepression or knockout of TBX5 enhancers delays transition from mid to late CM states. C_LIO_LIDeficient differentiation coincides with reduced expression of known cardiac genes. C_LI