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CRISPR/Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells
Jonathan M Geisinger1, Tim Stearns1,2
1Department of Biology, Stanford University, Stanford, CA 94305, USA.
Abstract:
While the mechanism of CRISPR/Cas9 cleavage is understood, the basis for the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus. Cell lines with functionally wild-type TP53 exhibited higher levels of cell cycle arrest compared to lines without. Chemical inhibition of TP53 protein combined with TP53 and RB1 transcript silencing alleviated induced arrest in TP53+/+ cells. Using dCas9, we determined this arrest is driven in part by Cas9 binding to DNA. Additionally, wild-type Cas9 induced fewer 53BP1 foci in TP53+/+ cells compared to TP53-/- cells and DD-Cas9, suggesting that differences in break sensing are responsible for cell cycle arrest variation. We conclude that CRISPR/Cas9 treatment induces a cell cycle arrest dependent on functional TP53 as well as Cas9 DNA binding and cleavage. Our findings suggest that transient inhibition of TP53 may increase genome editing recovery in primary and TP53+/+ cell lines.
Insights
CRISPR/Cas9 genome editing efficiency varies due to cell cycle arrest, influenced by TP53 status and Cas9 DNA interactions. Inhibiting TP53 may improve editing outcomes in certain cell types.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- CRISPR/Cas9 gene editing efficiency varies significantly across cell lines.
- The underlying reasons for this variation, particularly concerning DNA damage response and cell cycle, are not fully understood.
Purpose of the Study:
- To investigate the role of cell cycle progression and DNA damage response in mediating CRISPR/Cas9 editing efficiency variations.
- To identify factors contributing to differential mutant recovery in various human cell lines.
Main Methods:
- Utilized EdU incorporation to monitor cell cycle progression in response to CRISPR/Cas9 treatment.
- Analyzed the impact of TP53 status, TP53/RB1 transcript silencing, and Cas9 variants (dCas9, DD-Cas9) on cell cycle arrest.
- Quantified 53BP1 foci formation as a marker for DNA damage sensing.
Main Results:
- Human cell lines with functional TP53 exhibited increased cell cycle arrest post-CRISPR/Cas9 treatment compared to TP53-deficient lines.
- Chemical inhibition of TP53 and silencing of TP53/RB1 transcripts reduced cell cycle arrest in TP53+/+ cells.
- Cas9 binding to DNA, independent of cleavage, contributed to the induced cell cycle arrest.
- Wild-type Cas9 induced fewer 53BP1 foci in TP53+/+ cells than in TP53-/- cells, indicating differential DNA break sensing.
Conclusions:
- CRISPR/Cas9 treatment induces a TP53-dependent cell cycle arrest, influenced by Cas9 DNA binding and cleavage.
- Differences in DNA break sensing contribute to the observed variations in CRISPR/Cas9 editing recovery.
- Transient TP53 inhibition could potentially enhance genome editing recovery in specific cell types, including primary and TP53+/+ cells.
Related Concept Videos
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01:59CRISPR
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