Figure 1.

Generation of GPC3 knockout (KO) liver cancer cell lines using CRISPR-Cas9. A. Schematic representation of the genomic structure of GPC3 with the engineered exon site highlighted in red (Exon 3). The gRNA region (blue box) and PAM region (black box) were amplified using the GP1F and GP1R primer set, and the resulting DNA was sequenced. In HepG2-KO, a 13-nt deletion was observed, while in Hep3B-KO, a 7-nt deletion accompanied by a single nucleotide mutation (burgundy box) was identified compared to the wild type. B. Alignment of the amino acid sequences of partial human GPC3 and the two GPC3-KO proteins, presented in the one-letter code. Protein synthesis in the KO cells was terminated by a stop codon (indicated by a red-colored asterisk in the box). Open reading frames are highlighted in cyan, and amino acid sequences corresponding to the sgRNA-targeted regions are shown in bold letters.