Fig. 6: Mosaic genetic perturbation and scSeq in organoids highlights OTX2 regulome activity differences between retinal cell types.

a, Heat map shows OTX2 transcript expression, motif enrichment and positive regulome (+) and negative regulome (−) densities along the inner (I) to outer (O) and pseudotime axes from the reconstructed multimodal map. b, Transcriptome-based UMAP metacell plot showing OTX2 expression (left) or motif enrichment within accessible chromatin peaks (right). c, Global TF GRN highlighting OTX2 (black node) and the predicted positively (red) and negatively (blue) regulated genes within the inferred OTX2 regulon. d, Box plots show the distribution of OTX2 positively (top, +) or negatively (bottom, −) regulated targets within the GRN based on the expression correlation of each target to different retinal cell fates. e, Schematic of single-cell perturbation experiment using the CROP-seq method. Three gRNAs targeting OTX2 and four other TFs (Supplementary Information) were used together with a random non-targeting gRNA (dummy). Retinal organoids were infected with gRNA-containing lentiviruses at 19 weeks, and scRNA-seq and amplicon-seq were performed on suspensions at 22–24 weeks. f, UMAP projection colored by annotated cell type (left) or by cells with OTX2 (top right, red) or dummy (bottom right, dark gray) gRNAs detected. Gray cells represent unknown or other targeted TFs. g, Heat map showing gene expression modules (columns) and their activity in cell clusters (rows). Left sidebar shows cluster type. Bottom sidebar shows module clusters. Top side bar shows the differential module activity for the OTX2 gRNA cells relative to dummy control. h, Scatter plot showing the relationship between differential expression between OTX2 LOF and control (x axis) and predicted directionality in the OTX2 GRN targets (y axis). i, GO enrichments for modules that are significantly affected by OTX2 LOF. j, Heat map shows the module activity scores across the retinal organoid spatiotemporal multimodal map. CL, control; KO, knockout; Pt, pseudotime; wks, weeks; ER, endoplasmic reticulum.