Fig. 3: Multiplex single cell CRISPRa screening of regulatory elements in post-mitotic iPSC-derived neurons.
From: Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements
a Screen workflow. b gRNAs/cell. c Cells/gRNA. d UMAP projection of the neuron dataset from this study (blue, 51,183 cells downsampled to 5000 cells to aid with visualization) onto a sc-RNA-seq differentiation time-course from a similar differentiation protocol and NGN2 iPSC line (21,044 cells)29. e (Left) QQ-plot displaying observed vs. expected P-value distributions for targeting (blue) and NTC (downsampled) populations. (Right) QQ-plot for targeting tests against their intended/programmed target (blue) compared to targeting tests of all other genes with 1 Mb of each gRNA (pink) and NTCs (gray downsampled). P-values are from a two-tailed Wilcoxon rank-sum test. f Average log2 fold change and P-values exclusively for gRNAs that target putative enhancers in K562 cells (left) and iPSC-derived neurons (right). P-values as in panel (e). g (Top) Average log2 fold change in expression between cells with each targeting gRNA vs. controls for each of the primary/programmed target genes. (Bottom) Categorical heatmap showing which of the perturbations produced significant upregulation using an Empirical FDR approach (EFDR < 0.1). h Average log2 fold change between cells with a given gRNA and controls for select hit gRNAs (plotted as in Fig. 2d). Number of cells bearing each targeting gRNA (from left to right): CCND2 (n = 350), ZC3HAV1 (n = 765), TBR1 (n = 455), TBR1 (n = 655), BCL11A (n = 964), FOXP1 (n = 1198), FOXP1 (n = 917), TCF4 (n = 1051), TCF4 (n = 1253). Control cells are downsampled to have the same number of cells as the average number of cells detected per gRNA (n = 638) for visualization. P-values as in (e). i Of 14 targeted candidate promoters, five hit gRNAs for TCF4 target the same candidate promoter that aligns with biochemical marks of regulatory activity (ATAC-Seq and H3K27ac). Empirical P-values are visualized alongside tracks for iPSC-derived neuron ATAC-seq (accessibility)54, and H3K27ac54, and RefSeq validated transcripts (ENSEMBL/NCBI). P-values as in panel (e). EFDR sets as in panel (g). j Hits included multiple gRNAs targeting TBR1. Genomic tracks, P-values, and EFDR sets as in panel (i).
