Cas9 activates the p53 pathway and selects for p53-inactivating mutations
Open accessAbstract
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.
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In primary human hepatocytes, therapeutic concentrations of NTLA-2001 showed no evidence of previously described off-target mutagenesis mechanisms.
“In primary human hepatocytes, therapeutic concentrations of NTLA-2001 showed no evidence of previously described off-target mutagenesis mechanisms. 33,34”
From CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis (2021) · cites this paper as [34]
The paper’s Abstract (page not available in our copy) describes a previously observed Cas9-associated mechanism: the emergence and expansion of p53-inactivating mutations in human cancer cell lines. It does not test NTLA-2001 in primary human hepatocytes or establish that therapeutic concentrations show no evidence of that mechanism. (LLM, conf=0.86)
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- Authors
- Enache, O. M., Rendo, V., Abdusamad, M., Lam, D., Davison, D., Pal, S., Currimjee, N., Hess, J., Pantel, S., Nag, A., Thorner, A. R., Doench, J. G., Vazquez, F., Beroukhim, R., Golub, T. R., & Ben-David, U.
- Affiliations
- Broad Institute (US)
- Dana-Farber Cancer Institute (US)
- ORCID
- Venue
- Nature Genetics
- Published
- 2020-05-18
- Volume
- 52
- Issue
- 7
- Pages
- 662-668
- ISSN
- 1061-4036 (ISSN-L)
- 1546-1718
- PMC
- PMC7343612
- OpenAlex
- W3024591577
- Cited by
- 285
- References
- 38
- Citations by year
- 2026: 24 · 2025: 41 · 2024: 56 · 2023: 62 · 2022: 45 · 2021: 40 · 2020: 17
- FWCI
- 13.29
- Type
- journal article
- Language
- en
- Update notices
- correction (2020-06-25)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Genetics (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute on Drug Abuse (R01DA018828)
- National Cancer Institute (R01CA219943, R01CA215489, K08CA122833, R01CA188228)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Keywords
- Cas9, p53 pathway, TP53 mutation, CRISPR/Cas9 genome editing, off-target effects, DNA repair, DNA damage, cancer cell lines
- MeSH terms
- CRISPR-Associated Protein 9, DNA Mutational Analysis, Humans, Mutation, Streptococcus pyogenes, Transcription, Genetic, Tumor Suppressor Protein p53, Cell Line, Tumor, Metabolic Networks and Pathways, Transcriptome
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