We chose to test for Cas9-driven targeted mutagenesis on the
endogenous Trp53 gene in mouse embryonic fibroblasts (MEFs), a cell
line in which the biology of p53 has been thoroughly characterized and
where the gene is structurally intact.
Seventy-two hours post-transduction, cells were cultured in the
presence or absence of Nutlin-3a, a specific inhibitor of the MDM2–p53
interaction (Tovar et al. 2006). Cells were scored for the fraction of
GFP+ cells after 4 d of Nutlin-3a exposure using flow cytometry (Fig.
2B). Nutlin-3a potently activates a p53-dependent anti-proliferative
cellular response, which strongly and specifically selects for any
cells with disrupted Trp53 function (Efeyan et al. 2007). Much like
MLP-p53.1224 infected cells, the pQCiG-p53 GFP+ cells were rapidly
enriched for in the presence of Nutlin-3a, suggesting that
CRISPR-mediated gene disruption had initially occurred in at least a
proportion of the initial Cas9-expressing cells.