Fig. 5: Single-cell RNA-seq coupled with the dual-CRISPR screening system.
a, Illustration of adapting the dual-CRISPR screening system with scRNA-seq. Top: two distinct capture sequences (CS1 and CS2) were inserted into the stem loops of the guide RNA scaffolds. Bottom: the guide RNAs could then be captured together with the mRNA within individual single cells. Created with BioRender.com. b, Transcriptional changes of genes surrounding UCE PAX6_Ta measured by bulk RNA-seq. The y axis shows the log fold change of genes between the PAX6_Ta KO clone and control K562 cells. The x axis indicates the genomic coordinate. The dashed vertical line indicates the location of PAX6_Ta. Distinct colours represent different TADs. c, Transcriptional changes of genes surrounding PAX6_Ta measured by scRNA-seq. The y axis shows the log fold change of the captured genes between single cells with the guide RNA pair targeting PAX6_Ta and control K562 single cells. The x axis indicates the genomic coordinate. The dashed vertical line indicates the location of PAX6_Ta. Distinct colours represent different TADs. d, Top: epigenetic signatures surrounding enhancer E11:125334 as indicated by the red vertical line. Bottom: differential gene expression testing results. Violin plots show the normalized expression levels of candidate genes in perturbed (188 cells) and control (4,282 cells) groups. The gene EI24 was significantly downregulated (P = 0.0097, calculated using a MAST-fitted model).
