Journal Article

Cas9 activates the p53 pathway and selects for p53-inactivating mutations

Enache, Oana M., Lam, Daniel, Currimjee, Naomi, Doench, John G., Golub, Todd R., Nag, Anwesha, Beroukhim, Rameen, Thorner, Aaron R., Davison, Desiree, Pantel, Sasha, Vazquez, Francisca, Hess, Julian, Ben-David, Uri, Pal, Sangita, Abdusamad, Mai, Rendo, Veronica

2020

Overview

Cas9 is commonly introduced into cell lines to enable CRISPR–Cas9-mediated genome editing. Here, we studied the genetic and transcriptional consequences of Cas9 expression itself. Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon introduction of Cas9, specifically in wild-type TP53 ( TP53 -WT) cell lines. This was confirmed at the messenger RNA and protein levels. Moreover, elevated levels of DNA repair were observed in Cas9-expressing cell lines. Genetic characterization of 42 cell line pairs showed that introduction of Cas9 can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53 -WT and TP53 -null ( TP53 −/− ) cell lines. Lastly, Cas9 was less active in TP53 -WT than in TP53 -mutant cell lines, and Cas9-induced p53 pathway activation affected cellular sensitivity to both genetic and chemical perturbations. These findings may have broad implications for the proper use of CRISPR–Cas9-mediated genome editing. Cas9 expression induces DNA damage and activates the p53 pathway, and it can lead to the selection of cells with p53-inactivating mutations. Cas9 is less active in wild-type TP53 cell lines than in TP53- mutant cell lines.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/208 / 631/67 / Agriculture / Analysis / Animal Genetics and Genomics / Biomedical and Life Sciences / Biomedicine / Biotechnology / Cancer / Cancer Research / Cell Line, Tumor / CRISPR / CRISPR-Associated Protein 9 - metabolism / Deoxyribonucleic acid / DNA / DNA damage / DNA Mutational Analysis / DNA repair / Experiments / Gene expression / Gene Function / Genetic aspects / Genetic research / Genetic transcription / Genomes / Genomics / Human Genetics / Humans / Letter / Metabolic Networks and Pathways / mRNA / Mutation / p53 Protein / Proteins / RNA / Scientific equipment and supplies industry / Streptococcus pyogenes - enzymology / Transcription / Transcription, Genetic / Transcriptome / Tumor cell lines / Tumor proteins / Tumor Suppressor Protein p53 - metabolism