Figure 2

Construction of p53 knock-out PK15 cells using CRISPR/Cas9 genome editing system. A Schematic chromatogram representation of sgRNA targeting at the p53 genomic region. PAM sequences are underlined and highlighted in green. sgRNA targeting sites are highlighted in red. The red arrow indicates a putative cleavage site. The lower panel represents the functional domain of the p53 gene. The selected DNA oligos for the gRNA were annealed and cloned into the lentivirus vector for the CRISPR/Cas9 system. B Western blot analysis of the p53 expression after CRISPR/Cas9 lentivirus system targeting p53 locus infected PK15. The cells infected with lentiviruses containing gRNA-148 (148), gRNA-813 (813) or control gRNA (vector) were selected by puromycin, then puromycin-resistant cells and wild-type PK15 cells (WT) were analyzed using western blot. The data are mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 versus WT cells. C Sequencing of p53 locus amplified from the 148-gRNA (p53 ¹⁴⁸) and gRNA-813 (p53 ⁸¹³) generated cell clone. Red arrows indicate insertions or mutations. PAM sequences are highlighted in green. Red dashes indicate deleted bases or amino acids. Red characters indicate inserted base or mutated amino acids.