p53-pathway activation, inactivating TP53 mutations, and DNA damage were common with Cas9 expression.
Major Finding: p53 pathway activation, inactivating TP53 mutations, and DNA damage were common with Cas9 expression.
Concept: Experiments showed that genetic and chemical perturbation screens may be confounded by this effect.
Impact: These unanticipated sequelae of Cas9 expression should be considered when using CRISPR–Cas9 systems.
CRISPR–Cas9-mediated genome editing is now ubiquitous in biological research and may have clinical applications, but not all of the potential consequences of introducing Cas9 into cells are known. Enache, Rendo, and colleagues investigated the effects of inducing Cas9 expression in 165 human cancer cell lines and found substantial transcriptional differences between Cas9-expressing and wild-type (WT) cells. Specifically, 25 of the Cas9-expressing cell lines exhibited marked activation of the p53 pathway, and this effect was more common in cell lines with WT TP53 rather than TP53 harboring inactivating mutations. This observation, along with the well-established role of p53 in the DNA-damage response, raised the possibility that Cas9 induction could be associated with DNA damage. Indeed, Cas9 expression increased the number of DNA double-strand breaks in cells. In line with this finding, inactivating TP53 mutations emerged or expanded in cell lines expressing Cas9, as would be expected if p53 activity was a hindrance to stable Cas9 expression. In fact, TP53 landed in the top 4% of genes mutated in response to Cas9 expression. Additionally, a cell-competition assay revealed that expansion of cell populations harboring TP53-inactivating mutations was accelerated by Cas9 expression, an effect not seen with other tumor-suppressor genes. Demonstrating the functional relevance of these findings, genetic perturbation screens using CRISPR–Cas9-based gene editing or RNAi methods revealed notable discrepancies in TP53WT cell lines, and Cas9-expressing breast cancer cells were more sensitive to the MDM2 inhibitor nutlin-3 than WT cells. Thus, Cas9 expression may interfere with the interpretation of both genetic and chemical perturbation assays. In summary, this work demonstrates a previously unknown effect of Cas9 expression on p53, potentially producing consequences that should be considered in experiments and future therapeutics that employ CRISPR–Cas9-based techniques.
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