Identification of the TRRAP domain crucial for mutp53 stabilization and cell proliferation. (A) CRISPR-Cas9 screening of TRRAP protein domains. Namalwa-Cas9 cells were transduced in an arrayed format with 55 sgRNAs targeting different regions of TRRAP protein, covering all annotated domains according to Diaz-Santin et al.37 The proportion of transduced cells and the mutp53 level was quantified by flow cytometry. Negative cell selection (top, blue) and mutp53 depletion (bottom, red) is shown as fold depletion of transduced cells or mutp53 protein after 21 days or 7 days in culture, respectively. Every bar represents an independent sgRNA, and the location of each sgRNA relative to TRRAP protein is indicated along the x-axis. FAT, focal adhesion targeting domain; FATC, FRAP, ATM, TRRAP C-terminal domain; FRB, FKBP12-rapamycin binding domain; PI3K, phosphatidylinositol 3-kinase domain. (B) Comparison of fold depletion of cells (blue) and mutp53 protein (red) after transduction with sgRNAs targeting either domain X or any other region of TRRAP. P values were calculated with Student t test. Data reproduced from panel A. (C) p53 flow cytometry 7 days after TRRAP knockout in Namalwa-Cas9 cells. Values denote the ratio of the p53 MFI between transduced (red) and untransduced cells (gray). sgGFP, negative control; sgTP53, positive control. (D) Quantification of selection against sgTRRAP-transduced cells in BL-Cas9 cell lines. Bars indicate log2 fold depletion of sgRNA-transduced cells after 24 days in culture. The p53 status is specified for each cell line. sgGFP and sgmCherry, negative controls; sgPLK1, positive control. (E) Correlation of toxicity (depletion of sgRNA-transduced cells) and mutp53 depletion after TRRAP knockout using sgRNAs targeting different protein domains. Each data point represents a single sgRNA from panel A. Colors indicate the targeted domains. The Pearson correlation coefficient (r) and the corresponding P value are indicated.
