Fig. 4

Larger Cas13d library screening identified sorafenib-resistant circRNAs. a Number of gRNAs per circRNA in the library. b Schematic view of the drug selection screening. Cas13d lentivirus libraries were infected into CasRx stably expressed Huh7 cells then selected with puromycin treatment (time zero). Puromycin-resistant cells were further treated with DMSO (vehicle) or sorafenib for 14 days. Genomic DNA was extracted at day 14, and library representation was determined by deep sequencing. c Plot of fold-change of NT and circRNA gRNAs between DMSO- and sorafenib-treated samples. NT, non-targeting. d Scatterplot showing depletion of specific gRNAs after sorafenib treatment. Color dots indicate gRNAs corresponding with experimentally validated circRNAs as shown in Additional file 1: Figure S8f, g. e The robust rank aggregation (RRA) scores of top negatively selected circRNAs calculated by MAGeCK. Validated sorafenib-resistant circRNAs are labeled in color dots. CircRNAs are indicated with genomic locations and the host gene symbols at the end (e.g., chr12|112513481|112516545|NAA25|-|). f Relative expression levels of circCNIH4 and its parental mRNA upon knockdown of circCNIH4 by two independent gRNAs. g Knockdown of circCNIH4 sensitized Huh7 cells to sorafenib treatment. Knockdown of circCNIH4 significantly suppressed cell proliferation in the presence of sorafenib. Cell proliferation was measured by crystal violet staining, and absorbance at 590 nm of the treated cells from each group (day 6, day 8, day 10) was normalized to NT of the same day. NT, non-targeting. h Relative expression levels of circFMNL2 and its parental mRNA upon knockdown of circFMNL2 by two independent gRNAs. i Knockdown of circFMNL2 sensitized Huh7 cells to sorafenib treatment. Knockdown of circFMNL2 significantly suppressed cell proliferation in the presence of sorafenib. Cell proliferation was measured by crystal violet staining, and absorbance at 590 nm of the treated cells from each group (day 6, day 8, day 10) was normalized to NT of the same day. NT, non-targeting. j Relative expression levels of circCNIH4 and circFMNL2 in parental and sorafenib-resistant Huh7 cells. k Heatmap display of the relative expression levels of circCNIH4, circFMNL2 and their parental mRNAs upon knockdown of circCNIH4 and circFMNL2 by gRNAs in sorafenib-resistant Huh7 cells. l Knockdown of circCNIH4 or circFMNL2 sensitized sorafenib-resistant Huh7 cells to sorafenib treatment. Heatmap display of the relative cell proliferation of control and Cas13d-mediated circRNA-silenced Huh7 cells treated with either DMSO or sorafenib. Cell proliferation was measured by crystal violet staining, and absorbance at 590 nm of the treated cells from each group (day 6, day 8, day10) was normalized to NT of the same day. NT, non-targeting