Fig. 3
Systematic comparison of CRISPR-Cas13d and shRNA functional screenings for circRNAs. a Number of gRNAs and shRNAs per circRNA in the library. b Schematic view of the screenings. Cas13d and shRNA lentivirus libraries were infected into CasRx stably expressed Huh7 cells or naive Huh7 cells separately and selected by puromycin treatment (time zero). Puromycin-resistant cells were further cultured for 14 days. Genomic DNA was extracted at indicated time points and library representation was determined by deep sequencing. c Correlation heatmap showing the Pearson correlation coefficient between the levels of gRNAs/shRNAs in biological replicates of time zero samples (D0) and 14-day enrichment samples (D14) for Cas13d screen (left) and shRNA screen (right). d Gene set enrichment analysis (GSEA) revealed essential genes are enriched in negative selections for Cas13d screen (left) and shRNA screen (right). Essential genes serve as positive controls. The degree of enrichment is measured as normalized enrichment score (NES). e Scatterplots showing log2-transformed fold-change of gRNA/shRNA normalized read counts in D14 vs. D0 for Cas13d screen (left) and shRNA screen (right). Control, non-targeting controls; Essential, positive controls targeting known essential genes; Target, circRNAs highly expressed in HCC. f Scatterplots showing negatively selected gRNA/shRNAs and corresponding genes from Cas13d screen (left) and shRNA screen (right) with FDR < 0.25. CircRNAs are indicated with genomic locations and the host gene name at the end (e.g., chr10|32197099|32199491|ARHGAP12|). Positive controls only have gene names without genomic location (e.g., EIF4A3). g, h Top, relative expression levels of circZBTB44 and its parental mRNA upon knockdown of circZBTB44 by shRNAs (g) and gRNAs (h) in human Huh7 cells. Middle and bottom, proliferation rates of control and shRNA-mediated (g) or Cas13d-mediated (h) circZBTB44-silenced Huh7 cells. The number of cells was detected upon staining with crystal violet, and representative pictures are shown in the middle, while the proliferation curves are shown at the bottom. i, j Top, relative expression levels of circRHOT1 and its parental mRNA upon knockdown of circRHOT1 by gRNAs in Huh7 cells (i) or PLC/PRF/5 cells (j). Middle and bottom, proliferation rates of control and circRHOT1-silenced Huh7 cells (i) or PLC/PRF/5 cells (j). The number of cells was detected upon staining with crystal violet. The data shown are from one of two biological replicates with similar results, and error bars indicating the mean ± s.d. of three technical replicates. *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t test). ns, not significant