Figure 1.

CRISPR-mediated DNA damage enriches for cells with mutations in Trp53. A, Model describing how CRISPR and pharmacologic p53 activation used in the study are expected to affect targeted cells. B, Growth characteristics of Hox expanded bone marrow cells (from Cas9⁺ GFP⁺ mice), exposed to CRISPR (electroporated with a control or GFP targeting sgRNA; sgGFP), or pulsed for 8h with AMG232, or etoposide (ETOP). C, Kinetic analysis of apoptosis by flow cytometry-based TUNEL assay of Hox cells exposed to CRISPR (electroporated with sgGFP) or etoposide. D, Kinetic qPCR analysis of Cdkn1a expression of Hox cells exposed to CRISPR (electroporated with GFP sgRNA) or etoposide. E, Model describing experimental setup. F, WT and Trp53 KO Hox cells (Cas9⁺ and GFP⁺) were mixed and subjected to CRISPR [electroporated with sgGFP, or transduced with nontargeting ctrl (NTC) virus, sgGFP virus, CRISPR library virus], or an 8 hours pulse with etoposide or AMG232. After 7 days in culture, cells were sequenced, and the fraction of Trp53 KO sequences determined. G, WT and Trp53 KO Hox cells were mixed and cultured for seven days in a hypoxia chamber. H, WT and Trp53 KO B16 cells (Cas9⁺) were mixed, electroporated with control or a Ccr1 targeting sgRNA (sgCcr1), and injected subcutaneously into C57BL/6 mice. Day 21 tumors were collected and analyzed for Trp53 mutations. Data are shown as mean ± SEM, n = 3 (B–D), mean and individual values, n = 3 (F–H). Data are combined from three independently performed experiments (B–D and F–H). **, P < 0.01; ***, P < 0.001; n.s., nonsignificant by two-way ANOVA and Tukey posttest (B–D), one-way ANOVA and Tukey posttest (F–H).