4.8 Article

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

Journal

NATURE GENETICS
Volume 52, Issue 7, Pages 662-+

Publisher

NATURE PORTFOLIO
DOI: 10.1038/s41588-020-0623-4

Keywords

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Funding

  1. NIH [R01 CA18828, CA215489, CA219943]
  2. Gray Matters Brain Cancer Foundation
  3. Pediatric Brain Tumor Foundation
  4. HHMI
  5. HFSP
  6. Azrieli Foundation
  7. Richard Eimert Research Fund on Solid Tumors
  8. Tel-Aviv University Cancer Biology Research Center
  9. Israel Cancer Association

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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.

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