Fig. 1: CRISPR/Cas9 screen identifies Naa60 as mediator of Pt drug sensitivity in BRCA1/2;p53-deficient mouse mammary tumor cells.
From: NAA60 facilitates LRRC8A- and LRRC8D-mediated platinum drug uptake
a CRISPR/Cas9 screen layout, where KB2P (K14cre;Brca2F/F;Trp53F/F) mouse mammary tumor cells were transduced (1) using the GeCKo V2 pool B library and subsequently selected using 0.2 µM or 0.25 µM of cisplatin over the course of 7 days (2). gDNA was harvested, sequenced and subjected to enrichment analysis using MAGeCK (3). The top hits were verified using CRISPR/Cas9 mediated gene knockout (4). Created in BioRender. Widmer (2025) https://BioRender.com/ynvrayk. b, c Volcano plots of the results from screens performed with either 0.2 µM or 0.25 µM cisplatin over the course of 7 days. Size of the points was scaled to −log10 of FDR. d Representative big gene deletion knockout PCR control of wild-type (ntgB1 and ntgC1) and monoclonal Naa60 knockout KB1PM5 cell lines (Naa60_24A4, Naa60_12A6, and Naa60_13C3) used in the validation experiments. e, f RTqPCR and gel electrophoresis of the resulting product of control and Naa60 knockout lines. g, j Clonogenic survival assays of KB1PM5 wild-type and NAA60-deficient cell lines treated with cisplatin or carboplatin for 24 h with the indicated drug concentrations. Representative images of selected lines and concentrations are shown. h, k Quantification of clonogenic growth assays in the presence of cisplatin or carboplatin. Data represent mean ± SD of five independent replicates for cisplatin and three independent replicates for carboplatin and were fitted to a non-linear regression dose-response curve (log(inhibitor) vs. normalized response -Variable slope). p values are calculated by one-way ANOVA followed by Tukey’s multiple comparisons test for the log(IC50) values of the survival curves. Kaplan–Meyer overall survival curves of mice transplanted with NAA60-deficient (Naa60_24A4) or wild-type control (ntg) KB1PM5 organoids treated with either vehicle or 6 mg/kg of cisplatin for (i) and 50 mg/kg of carboplatin for (l). N = 5 mice per group. Statistical analysis was performed with the log-rank test (Mantel–Cox).
