CRISPR/Cas9-mediated disruption of Oncopig KRASG12D and TP53R167H transgenes. (A) Schematic representation of the Oncopig transgene showing gRNA target sites and primers used for PCR. IRES, Internal ribosome entry site. (B) KRASG12D and TP53R167H editing efficiencies at multiple time points post transfection with Cas9 and gRNAs. (C) Frameshift mutations resulting in protein truncation for 2 Oncopig TP53R167H KO HCC cell lines developed via single cell clone isolation and screening. Dashed line marks the cleavage position, and dashed grey boxes represent nucleotide deletions. Dotted regions represent frameshifts in predicted protein sequences. (D) Positive arginase-1 staining (brown) of parental and TP53R167H KO cell lines (scale bar, 300 μm). (E) Cellular proliferation of Oncopig parental and TP53R167H KO HCC cell lines. Values represent mean ± S. D. (n ? 3). **indicates P < 0.001.