Fig. 5: SUM-seq paired with CRISPRi/a characterizes the roles of GATA2, NR4A2 and SOX17 on regulatory network dynamics along spontaneous hiPS cell differentiation. | Nature Methods

Fig. 5: SUM-seq paired with CRISPRi/a characterizes the roles of GATA2, NR4A2 and SOX17 on regulatory network dynamics along spontaneous hiPS cell differentiation.

From: Single-cell ultra-high-throughput multiplexed chromatin and RNA profiling reveals gene regulatory dynamics

Fig. 5: SUM-seq paired with CRISPRi/a characterizes the roles of GATA2, NR4A2 and SOX17 on regulatory network dynamics along spontaneous hiPS cell differentiation.

a, A schematic overview of the 54-plex arrayed CRISPRi/a screens targeting lineage TFs GATA2, NR4A2 and SOX17 along a time course of spontaneous differentiation (n = 2 clones per CRISPR modality; 1–2 gRNAs per target and nontargeting control (NT-ctrl)). Created with BioRender.com. b,c, WNN UMAP of the CRISPRi/a SUM-seq screens along spontaneous differentiation of EBs (b) or within a monolayer (c) colored by the combination of their cluster and perturbation (sample index; collapsed by targeted gene). d,e, UMAP representation of GRNs inferred for CRISPRi (d) and CRISPRa (e) cells. GRN TFs are colored by the multiplication of their activity (AUCell) and pseudotime across all cells (approximating the pseudotime point of their highest activity), while shape corresponds to the day of the highest regulon AUCell score. The line thickness indicates the number of shared targets of regulon genes. f, Top: UMAP of CRISPRi cells colored by pseudotime and overlaid with contour lines indicating the location of GATA2-KD (dashed lines) and control cells (solid lines) from day 4 to 18. Bottom: UMAPs showing higher AUCell scores for the GRHL1 regulon in the GATA2-KD trajectory (left) and SOX9 regulon in the control trajectory (right). g, Gene Ontology enrichment (hypergeometric test) for differentially expressed genes between the CRIPSRi control and GATA2-KD trajectories. Size represents gene ratio and color the −log10 FDR-corrected P value. h, Top: UMAP of CRISPRa cells colored by pseudotime and overlaid with contour lines indicating the location of NR4A2-OE (dashed lines) and control (solid lines) cells from day 12 to 18. Bottom left: bar plot depicting the fraction of control, NR4A2-OE, SOX17-OE and GATA2-OE cells in the two day 18 subpopulations. Bottom right: UMAP representation of GRNs inferred for day 18 of the CRISPRa screen. GRN TFs are colored by the log2 fold change in regulon AUCell score between the NR4A2-OE and control groups. Shape corresponds to the day of highest regulon AUCell score. i, Gene Ontology and WikiPathways enrichment (hypergeometric test) for differentially expressed genes for NR4A2-OE, SOX17-OE and control groups of cells in the day 18-1 subpopulation. Size represents gene ratio and color the −log10 FDR-corrected P value. Pos., positive; neg., negative; reg., regulation.

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