Fig. 1

Generation of YB-1 knockout cancer stem cells using the CRISPR/Cas9 system. a Schematic representation of the guide RNA targeting YB-1 gene. The sequence of PAM (protospacer adjacent motif) was boxed. b T7 endonuclease 1 (T7E1) assay. Melanoma stem cells were transfected with pHBCas9/gRNA-Pure plasmid containing YB-1 gRNA. As a control, the vector only (without YB-1 gRNA) was included in the transfection. The DNA containing target site of YB-1 gRNA was amplified by PCR using the genomic DNA extracted from the transfected cells. The PCR product was digested with T7E1, followed by agarose gel electrophoresis. The experiments were biologically repeated for three times. M, DNA marker. c The sequencing of YB-1 mutant of cancer stem cells. The YB-1 gene was amplified from YB-1-mutated melanoma stem cells (MDA-MB-435) or breast cancer stem cells (MCF-7) and then sequenced. The sequences of two YB-1 alleles of mutants were indicated. The deletion mutations were shown with dashed boxes and the dot mutation was circled. d Western blot analysis of YB-1 in YB-1 mutants of cancer stem cells. β-tubulin was used as a control. The representative images of three experiments were shown