Fig. 5: CRISPR-modified in vitro models, with EGFR mutation and oncogenic fusions.
From: Genomic and biological study of fusion genes as resistance mechanisms to EGFR inhibitors
a The structures of the fusion oncogene and the location of designed single guide RNAs (sgRNAs) are shown, with representative sequencing chromatograms of fusion cDNA derived from bulk CRISPR-modified EGFR mutant PC-9 cells. e: exon; UTR: untranslated region. b Colony formation assay after 1 week of treatment with osimertinib, using parental PC-9 cells or CRISPR-modified PC-9 cells that express fusion oncogenes. c Breakpoints of CCDC6-RET fusion in bulk CRISPR-modified PC-9 cells. d Expression of RET protein in permeabilized parental or CRISPR-modified PC-9 models, evaluated with use of flowcytometry. PC-9CCDC6-RET bulk cells were selected with 100 nM osimertinib for 1 week, and then a single clone was picked. Ratio of RET-expressing cells in each of four categories are indicated. Pseudo-color represents cellular density. e Results of cell viability assay after 72 h of osimertinib treatment. Half maximal inhibitory concentrations (IC50s) are shown for parental PC-9 cells and for single clones from CRISPR-modified PC-9CCDC6-RET models, selected with or without 1 week of exposure to 100 nM osimertinib (n = 3 biological replicates, mean ± s.d.). f Knockdown of RET, BRAF, FGFR3, or ALK genes in CRISPR-modified PC-9 clones after 48 hours of siRNA treatment, is shown by western blot analyses. WT: wild type. g Knockdown of RET, BRAF, FGFR3, or ALK genes by siRNA in CRISPR-modified PC-9 cells resensitized them to 1 μM osimertinib (n = 3 biological replicates, mean ± s.d., two-sided t test, **p < 0.01). Source data of e–g are provided as a Source Data file.
