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. 2023 Apr 25;12:e86206. doi: 10.7554/eLife.86206

Figure 1. Single-cell screens of congenital heart defect (CHD)-associated enhancers during cardiomyocyte (CM) differentiation.

(A) Genome browser snapshot emphasizing features of a targeted enhancer about 15 kb upstream of the RBM20 gene. Yellow highlights enhancer region. (B) H3K27ac and open chromatin (ATAC-Seq) enrichment for targeted enhancers across multiple time points of CM differentiation (Liu et al., 2017; Tompkins et al., 2016; Zhang et al., 2019). (C) Expression of putative target genes across multiple stages of CM differentiation. Expression defined as fold change over the day 0 expression of a target gene (Tompkins et al., 2016). (D) Schematic of single-cell CRISPRi screen. H9-dCas9-KRAB cells are infected with a lentiviral sgRNA library and differentiated over 8 days into CMs followed by scRNA-seq. Individual cells are linked to sgRNA perturbations and changes in transcriptional cell state. (E) UMAP visualization of H9-derived cells after 8 days of differentiation. Seven Louvain clusters indicated. (F) Expression of the top 100 cluster defining genes for each Louvain cluster cell type. (G) MALAT1 expression in control (non-targeting) and sgMALAT1 cells (*p<0.05 by Mann-Whitney U). (H) Distribution of cells receiving sgNT, sgTBX5 PROM1, sgTBX5 ENH6 that differentiate into CM and mesoderm states (*p<0.05 and **p<0.001 by hypergeometric test).

Figure 1.

Figure 1—figure supplement 1. Single-cell CRISPRi screen validation and statistics.

Figure 1—figure supplement 1.

(A) Brightfield images of H9-dCas9-KRAB cells receiving sgRNAs targeting OCT4 or NT control (Scale bar = 200μm). (B) (Top) OCT4 expression in cells receiving sgRNAs targeting OCT4 or NT control. For both, qPCR quantification normalized to reference gene (RPLP0) and then compared with control cells. (Bottom) MALAT1 expression in cells receiving sgRNAs targeting MALAT1 or NT control (n = 3 technical replicates). (C) Distribution of sgRNA counts in cells with sgRNAs detected. (D) Distribution of cell counts for each sgRNA. (E) Distribution of cell counts for each targeted region. (F) Feature plots of marker genes for neuronal (+SOX2), cardiomyocyte (+TNNT2), mesoderm (+FN1), epithelial (+EPCAM), cardiac fibroblast (+COL3A1), and endoderm cells (+TTR). (G) NGFRAP1 expression in cells receiving sgRNA targeting NGFRAP1 or NT controls (*p<0.05 by Mann-Whitney U). (H) Distribution of cells receiving sgRNAs targeting ZIC2 promoter or NT controls after differentiation (*p<0.05 by hypergeometric test). (I) TBX5 expression in cells receiving sgRNAs targeting TBX5 enhancer 3, enhancer 5, or non-targeting NT control (n = 3 technical replicates).