Extended Data Fig. 8: Introduction of KRASG12D mutation on triple-pathway mutant organoids. | Nature Cancer

Extended Data Fig. 8: Introduction of KRASG12D mutation on triple-pathway mutant organoids.

From: Recapitulating the adenoma–carcinoma sequence by selection of four spontaneous oncogenic mutations in mismatch-repair-deficient human colon organoids

Extended Data Fig. 8

(a) Triple-pathway mutant organoids were transfected with Cas9, sgRNA and the oligonucleotide previously designed for KRASG12D mutation. KRASG12D mutants were selected in the medium lacking EGF, with the EGFR inhibitor gefitinib (-WRNE+Nut3+Gef). Small single organoids survived and grew (Inset). Representative pictures from n = 3 clonal organoid lines. Scale bars, 500 µm. (b) Picked up surviving KRASG12D mutant organoid (representative pictures from n = 3 clonal organoid lines). Scale bars, 100 µm. (c) Single survived organoids were expanded and passaged in the medium lacking EGF, with the EGFR inhibitor gefitinib (-WRNE+Nut3+Gef). Representative pictures from n = 3 independent replicates. Scale bars, 500 µm. (d) Sequence analysis of the targeted KRAS exon. Oncogenic GGT > GAT mutation is indicated in green; silent mutations are in blue; protospacer adjacent motif (PAM) is underlined in red.

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