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Fig. 5 Figure 5. Optimization of scGESTALT Lineage Recorder for Better Barcode Recovery (A) Schematic overview of CRISPR-Cas9 lineage recording. Optimized scGESTALT comprises a barcode cassette in the 3′end of DsRed transgene (single copy) and the medaka beta-actin promoter. Embryos were injected with Cas9 protein and DsRed sgRNAs, and animals were profiled at 15 dpf by scRNA-seq. (B) Pairwise comparisons using cosine dissimilarity of barcode edit patterns from four (ZF1–ZF4) edited 15 dpf larval brains. (C) Chord diagram of the nature and frequency of deletions within and between target sites. Each colored sector represents a target site. Links between target sites represent inter-site deletions; self-links represent intra-site deletions. Link widths are proportional to the edit frequencies. (D) Type of edit at each target site within the barcode from edited ZF1–ZF4 larval brains. (E) Heatmap of lineage relationships between non-retinal and retinal cell types in the eye. All clusters with >3 cells and all barcodes with >1 cell were used to determine if there is enrichment of cell-type-specific barcodes across each cluster pair. Blue indicates significant enrichment and lineage segregation. Purple indicates no significant enrichment and no lineage segregation. Grey indicates insufficient sampling power and undefined lineage status. Cluster numbers are indicated (e.g., C45) and either cell type gene markers (e.g., cldna+) or the exact name of the cell type (e.g., cone bipolar cells) are indicated along the rows. Along the columns, the numbers within the brackets indicate the number of barcodes and number of cells, respectively, for that cluster. (F) Heatmap of lineage relationships between brain regions and the retina. Neuron clusters that could be pseudospatially assigned to each region were used (see Table S1). Analysis, layout, and color code are same as in (E). (G) Heatmap of lineage relationships between neuronal cell types in the forebrain and midbrain. Analysis, layout, and color code are same as in (E). Clusters were assigned to a brain region (e.g., pallium, hypothalamus), and for clusters where a more precise location could not be inferred, a gene marker is indicated (e.g., pitx2+). (H) Heatmap of lineage relationships between brain progenitor clusters. Analysis, layout, and color code is same as in (E). Cell type marker genes are indicated along with the cluster number. URL, upper rhombic lip (I) Bar plot of the proportion (based on Jaccard Index) of granule cell (cerebellum neurons) barcodes that are shared with each brain progenitor cluster. Cluster numbers are the same as in (H).