Fig. 6: Single-cell CRISPR & transcriptome profiling of myoblast fusion screen hits.

a Schematic of the experimental design. The CROP-seq, a lentiviral gRNA construct, was utilized to express regular gRNAs (for knockout) and polyadenylated gRNAs (for scRNA-seq detection) targeting 250 myoblast fusion screen hits. This enabled simultaneous scRNA-seq, CRISPR knockout & gRNA detection. b’ Force-directed graph layout of 10 cell clusters. Marker genes for each cluster was provided in Supplementary Data 8. b” UMAP plot illustrating the expression of myoblast markers (MKI67, CCND1, BUB1, CENPF, TOP2A) in cluster #4 and Myomaker & muscle structural genes which are predominantly expressed in DM clusters (#1, #2, #5, #7). b”’ Contour density plots of cells assigned to MYMK and MyoD gRNAs in UMAP space. c GO analysis of top differentially expressed (DE) genes in cluster #4 versus #1/2/5/7, collectively referred to as “myogenesis signature genes” (see also Supplementary Data 9). Prolif: proliferation; Diff.: muscle differentiation. Statistical analysis was performed using the clusterProfiler in R. d Heatmap displaying the median relative expression (column z-score) of myogenesis signature genes (columns), aggregated across cells expressing gRNAs targeting genes from the same protein complex or pathway (rows). e Distribution of myogenesis signature intensity across single cells (left) and the number of cells (right) for each pathway/protein complex. Black dots indicate the median. f Heatmap of differentially expressed marker genes in each cluster. A few top up-regulated genes for each cluster are shown. Genes upregulated in multiple clusters are assigned to the cluster with the highest fold change. To the right of the heatmap, for each cluster, are (left to right): top marker genes, top GO terms representing cell makers, overrepresented sgRNAs by odds ratio, and the assigned cluster name.