Extended Data Fig. 4: Impact of individual sgRNAs on cell viability and drug response in the base editing drug resistance screens in MCF10A cells.

(a) Scatter plot comparing the individual sgRNA LFCs between plasmid and day 19 and between nontreated and TKI-treated samples. Negative-log FDR values for drug resistance sgRNAs are shown. Dashed squares represent the hit inclusion criteria: LFC (plasmid vs day 19) > −1.5 and LFC (nontreated vs TKI-treated) > 0.75. (b) TKI response validation of drug resistance screen hits in MCF10A cells. Relative viabilities of edited and unedited MCF10A cells after gefitinib or osimertinib treatment as measured by the CellTiter-Glo assay. Cells were infected in triplicates and treated for 5 days with 0, 0.15, 0.3, 0.6, 1.2 or 2.4 μM of the respective drug before viability measurements. Solid lines represent replicate means and shaded areas represent the standard error. (c) Deep sequencing data for individual sgRNA validation in MCF10A cells. Cells were infected with the indicated sgRNA and base editor pairs in three biological replicates (n = 3) and later split into control and treatment arms with gefitinib or osimertinib. The percentages of reads corresponding to the four most represented alleles at each target site are shown for nontreated and drug-treated cells. Nucleotide sequences represent the coding DNA strand and black squares denote wild-type EGFR sequences. Holm-adjusted P values are shown for two-sided t-tests. (d) Diagram showing combined ABE8e and BE3.9max screen hits along the EGFR tyrosine kinase and C-terminal domains for cells treated with gefitinib or osimertinib. Hits are defined as sgRNAs with LFC > −1.5 between plasmid and day 19 and LFC > 0.75 between nontreated and TKI-treated cells. Notable post-translational modifications and intramolecular interactions are shown. (*) Validation of the Lys852Gly [ABE8e] sgRNA showed that the main variant conferring drug resistance was Lys852Glu (Extended Data Fig. 5d).