Generation and validation of isogenic, WT, and p53-null E1A;Hras^G12V;H11^Cas9 MEFs. (A) Trp53 exon structure and the binding sequences of the two sgRNAs used to target p53 in this study. PAM motifs are in red. Exons encoding different p53 domains are also indicated: transactivation domain (TAD), proline rich domain (PR), DNA binding domain (DBD), nuclear localization signal (NLS), and oligomerization domain (OD). (B) Summary of ICE analysis on E1A;Hras^G12V;H11^Cas9 MEFs used in this study. ICE score represents the percentage of sequences within the pool with non–WT sequences, whereas the KO score represents the percentage of sequences in the pool with either a frameshift or 21+-bp indel. R squared values refer to the Pearson correlation coefficient for the ICE score. (C) Western blot analysis of p53, p21, and Mdm2 after doxorubicin (dox) treatment of all nine E1A;Hras^G12V MEF lines used in this study. Gapdh serves as a loading control. (D) qRT-PCR analysis of p53 target gene expression in all nine E1A;Hras^G12V MEF lines used in this study. n = 3 cell lines/sgRNA (in triplicate). Data are presented as mean ± SD, after normalization to β-actin. (E) Representative images of soft agar assays of all nine sgRNA-targeted E1A;Hras^G12V MEF lines used in study. n = 3 cell lines/sgRNA. Scale bar, 3.5 mm. (F) Cell viability analysis (AnnexinV, PI-negative cells) on all sgRNA-targeted E1A;Hras^G12V MEFs 24 h after DNA damage (doxorubicin treatment). n = 3 cell lines/sgRNA, three to five independent experiments. Data are presented as mean ± SD. For A–F, all experiments were performed in 5% O₂.