Cas9 activates p53 and selects for p53-inactivating mutations

Cas9 is commonly introduced into cell lines to enable CRISPR-Cas9 genome editing. Off-targets effects and genotoxicity have been associated with prolonged Cas9 activity in the presence of guide RNAs, but the cellular consequences of Cas9 expression per se still remain unknown. We performed extensive transcriptional and genetic characterization of cancer cell lines before and after Cas9 introduction. Gene expression profiling of 165 human cancer cell lines and their Cas9 derivatives revealed upregulation of the p53 pathway in 48.3% of the TP53-WT cell lines. Immunoblotting and RT-qPCR confirmed a significant upregulation of multiple p53 transcriptional targets involved in the detection of DNA damage, regulation of cellular stress and induction of apoptotic response. To determine whether Cas9-induced p53 activation occurred in response to DNA damage, we analyzed DNA damage-associated transcriptional signatures, and measured DNA damage directly by γ-H2AX foci quantification. Both assays confirmed a significant increase in DNA damage following Cas9 introduction. Deep targeted sequencing of 450 cancer-related genes in 42 cell line pairs demonstrated that Cas9 introduction could lead to the emergence and expansion of p53-inactivating mutations, in ~7.5% of the cell lines. These findings were validated prospectively in competition experiments with isogenic TP53-WT/TP53-null cell lines. Lastly, we compared Cas9 activity across 719 human cell lines and found that Cas9 was significantly less active in TP53-WT cell lines, confirming that p53 is a barrier to the efficient expression of Cas9 in human cells. These findings have important implications for the use of CRISPR-Cas9 in basic science and for its clinical applications.