CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based
Long Jiang1, Katrine Ingelshed2, Yunbing Shen1
1Department of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden.
Abstract:
Inactivating p53 mutations are the most abundant genetic alterations found in cancer. Here we show that CRISPR/Cas9-induced double-stranded DNA breaks enrich for cells deficient in p53 and in genes of a core CRISPR-p53 tumor suppressor interactome. Such enrichment could predispose to cancer development and thus pose a challenge for clinical CRISPR use. Transient p53 inhibition could suppress the enrichment of cells with these mutations. The level of DNA damage response induced by an sgRNA influenced the enrichment of p53-deficient cells and could be a relevant parameter in sgRNA design to limit cellular enrichment. Furthermore, a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, these data provide details about p53 biology in the context of CRISPR-induced DNA damage and identify strategies to enable safer CRISPR use. SIGNIFICANCE: CRISPR-mediated DNA damage enriches for cells with escape mutations in a core CRISPR-p53 interactome, which can be suppressed by transient inhibition of p53.
Insights
CRISPR/Cas9 DNA breaks can increase cancer-driving p53 mutations. Transiently inhibiting p53 or optimizing sgRNA design can prevent this enrichment, enabling safer clinical CRISPR applications.
Area of Science:
- Cancer Biology
- Gene Editing Technologies
- Tumor Suppressor Pathways
Background:
- Inactivating p53 mutations are common in cancer.
- CRISPR/Cas9 technology is a powerful tool for gene editing.
Purpose of the Study:
- To investigate how CRISPR/Cas9-induced DNA damage affects cells with p53 mutations.
- To identify strategies for mitigating the enrichment of p53-deficient cells during CRISPR use.
Main Methods:
- Utilized CRISPR/Cas9 to induce double-stranded DNA breaks.
- Analyzed enrichment of p53-deficient cells and interactome genes.
- Assessed the impact of transient p53 inhibition and sgRNA design.
- Examined a dataset of over 800 human cancer cell lines.
Main Results:
- CRISPR/Cas9 breaks enrich for cells deficient in p53 and its interactome.
- This enrichment poses a challenge for clinical CRISPR applications.
- Transient p53 inhibition suppressed the enrichment of p53-mutated cells.
- DNA damage response levels influenced p53-deficient cell enrichment.
- Baseline CDKN1A expression predicts an active CRISPR-p53 axis.
Conclusions:
- CRISPR-induced DNA damage can select for escape mutations in the p53 pathway.
- Strategies like transient p53 inhibition and informed sgRNA design can enhance CRISPR safety.
- Understanding p53 biology is crucial for the clinical translation of CRISPR technology.
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