Inhibition of p53 improves CRISPR/Cas-mediated precision genome editing
Emma Maria HaapaniemiiD, Sandeep Kumar BotlaiD, Jenna PerssoniD, Bernhard SchmiereriD, Jussi TaipaleiD
Abstract
We report here that genome editing by CRISPR/Cas9 induces a p53-mediated DNA damage response and cell cycle arrest. Transient inhibition of p53 prevents this response, and increases the rate of homologous recombination more than five-fold. This provides a way to improve precision genome editing of normal cells, but warrants caution in using CRISPR for human therapies until the mechanism of the activation of p53 is elucidated.
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In-depth summary
Claude Sonnet 4.6 · xhigh effort · 7 Nov 2026 · from the full text of version 1 (25 Aug 2017) · CC BY 4.0
Cas9-induced double-strand breaks trigger p53/p21/RB1-dependent G1 arrest in RPE-1 cells; transient p53 inhibition via MDM2 markedly raises homologous recombination precision editing efficiency.
Normal and primary cells are hard to edit precisely because they favor error-prone repair or die, and earlier fixes (more template, NHEJ inhibitors, better transfection) gave gains of up to threefold without a mechanism. This work identifies a p53-dependent DNA damage response as an explanation, and one that also confounds dropout CRISPR screens.
Key results
ShownIn genome-wide CRISPR dropout screens in human RPE-1 cells, p53 status determined whether essential genes could be detected; gRNAs against p53, p21 and RB1 were strongly enriched in p53 wild-type cells.
Median rank of ribosomal gene gRNAs was 8,034 in p53+/+ (random depletion) vs 1,627 in p53-/- (lowest decile). p21 guides enriched 4.7 fold in p53+/+ vs 0.8 fold in p53-/-. Brunello library; GSEA found essential pathways only in p53-/-. · Main text; Figure legends
ShownBrief, non-integrated Cas9 RNP exposure was enough to activate p53 signaling and G1 arrest in p53 wild-type RPE-1 cells but not in p53-null cells, without a detectable rise in apoptosis.
RNP targeting the RNF1 locus raised p21 and the G1 fraction in p53+/+ cells (n=4, p<0.05). Cleaved Caspase 3 at 24h was not increased in p53+/+ (n=4). All readouts by flow cytometry. · Main text; Figure legends
ShownSupplying the p53 antagonist MDM2 raised homologous recombination repair of a mutated GFP reporter in a dose-dependent way in p53 wild-type RPE-1 cells, indicating that the DNA damage response limits precision editing.
GFP-positive percentage after RNP plus ssDNA repair template increased dose-dependently with MDM2 (n=6 per condition); abstract reports an HR increase of more than five-fold with transient p53 inhibition. · Abstract; Main text; Figure legends
SuggestedInhibitors of type I interferon signaling branches did not improve precision editing in the GFP reporter line, so interferon response is probably not the main driver of Cas9 toxicity or G1 arrest in RPE-1 cells.
None of the tested inhibitors (Toll-like receptor, interferon-alpha, interleukin 1 beta pathways), alone or in combination, significantly changed editing efficiency (Supplementary Table S2, not shown in text). · Main text
ClaimedThe authors argue that editing selects for p53-deficient cells and that transient p53 inhibition has both benefits and tumorigenic risks, so therapeutic CRISPR use warrants caution.
Based on screen enrichment of p53 pathway guides and the discussion; no data on chromosomal rearrangements or tumorigenic outcomes after p53 inhibition are presented in the text. · Abstract; Main text
Methods, data and reproducibility
Genome-wide CRISPR/Cas9 dropout screens were compared in p53+/+ and p53-/- RPE-1 cells and analyzed with MAGeCK and gene set enrichment. RNP delivery assays and a GFP-repair reporter, read by flow cytometry, tested p53 response and the effect of MDM2 or interferon inhibitors.
- Genome-wide CRISPR/Cas9 dropout screens (Brunello library) in RPE-1 p53+/+ and p53-/- cells · human (RPE-1 cell line)
- RNP transfection (RNF1 locus) readouts of p21, G1 fraction and cleaved Caspase 3 in RPE-1 p53+/+ and p53-/- · human (RPE-1 cell line) · n = n=4
- RPE-1 p53+/+ mutant-GFP reporter line with RNP and ssDNA repair template, with MDM2 supplementation · human (RPE-1 cell line) · n = n=6 per condition
- Interferon pathway inhibitor tests in the RPE-1-GFP reporter line · human (RPE-1 cell line)
- Methods & tools
- CRISPR/Cas9, Cas9 ribonucleoprotein (RNP) delivery, Brunello gRNA library, MAGeCK (modified Robust Rank Aggregation), Gene set enrichment analysis, Flow cytometry, MDM2 (p53 antagonist), GFP repair reporter with ssDNA template, Cleaved Caspase 3 assay
- Reproducibility gaps
- Screen methods and Supplementary Tables S1 and S2 are referenced but not in the provided text; MCF10A data are 'not shown'; no code or sequencing data accessions are given.
Limitations (4)
- The mechanism by which Cas9 activates p53 is not elucidated, and the authors urge caution in therapeutic CRISPR use until it is understood. · stated by the authors
- Inhibiting p53 leaves cells transiently vulnerable to chromosomal rearrangements and other tumorigenic mutations, and editing itself favors growth of p53-deficient cells. · stated by the authors
- Functional editing tests used one immortalized line (RPE-1) and a GFP reporter with MDM2; primary cells, endogenous loci and indel-versus-precise-edit sequencing are not reported, and MCF10A data are not shown. · noted in this summary
- The text reports no fold change or statistics for the MDM2 dose response beyond n=6; the five-fold figure appears only in the abstract. · noted in this summary
Who should read:Read this if you run CRISPR dropout screens or precision editing in p53-proficient normal cells and want evidence on p53-driven G1 arrest and its inhibition.
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