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. 2019 Nov 19;8:e50223. doi: 10.7554/eLife.50223

Figure 1. Genome-wide CRISPR-Cas9 screening identifies determinants of EGFR-TKI sensitivity in EGFR-mutant NSCLC.

(A) Cell viability assessment by CellTiter-Glo assay of HCC827 cells treated with serial dilutions of erlotinib for 72 hr. Error bars represent mean ± standard deviation (SD); n = 4. (B) Kinetic cell proliferation assay monitored by IncuCyte for HCC827 cells cultured in the presence of DMSO control or 1 µM erlotinib over a 30 day period. (C) Crystal violet staining colony formation assay of HCC827 cells treated with DMSO or 1 µM erlotinib for the indicated days. (D) Schematic outline of the genome-wide CRISPR-Cas9 screening workflow in HCC827 cells. (E) Scatterplot depicting gene level results for erlotinib negatively selected hits in the CRISPR screen. A number of representative hits are shown in color. (F) Scatterplot depicting gene level results for erlotinib positively selected hits in the CRISPR screen. A number of representative hits are shown in color. (G) STRING protein network of the 35 negatively selected hits as defined in (E). The nodes represent indicated proteins, and colored nodes highlight proteins enriched in certain signaling pathways. The edges represent protein-protein associations, and the line thickness indicates the strength of data support. The minimum required interaction score was set to default medium confidence (0.4), and the disconnected nodes were removed from the network. (H) STRING protein network of the 47 positively selected hits as defined in (F).

Figure 1.

Figure 1—figure supplement 1. CRISPR-Cas9 screening reveals genetic determinants of EGFR-TKI sensitivity.

Figure 1—figure supplement 1.

(A) Cumulative frequency of sgRNAs in the library plasmid and after 21 days of DMSO or erlotinib treatment in HCC827 cells. (B) Box plot showing the distribution of sgRNA representations in the library plasmid and after 21 days of DMSO or erlotinib treatment in HCC827 cells. (C) Scatterplot showing the comparison of sgRNA frequency between DMSO and erlotinib treated HCC827 cells. (D) Dot plot showing the distribution of individual sgRNAs targeting erlotinib negatively selected hits in the CRISPR screen. Data are presented as log2 fold change of each sgRNA sequence based on the abundance in the erlotinib-treated versus DMSO-treated cell population. (E) Dot plot showing the distribution of individual sgRNAs targeting erlotinib positively selected hits in the CRISPR screen. Data are presented as log2 fold change of each sgRNA sequence based on the abundance in the erlotinib-treated versus DMSO-treated cell population. (F) Reactome pathway analysis for erlotinib negatively selected hits in the CRISPR screen. (G) Reactome pathway analysis for erlotinib positively selected hits in the CRISPR screen.