p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells

Area of Science:

• Molecular Biology
• Genetics
• Stem Cell Biology

Background:

• CRISPR/Cas9 technology enables genome engineering and screening in human cells.
• Engineering human pluripotent stem cells (hPSCs) with CRISPR/Cas9 has historically shown reduced efficiency compared to other cell types.
• Previous studies underestimated Cas9 toxicity in hPSCs due to low transfection and double-strand break (DSB) induction rates.

Purpose of the Study:

• To investigate the efficiency and challenges of CRISPR/Cas9 genome engineering in hPSCs.
• To identify the underlying mechanisms responsible for reduced genome engineering efficiency in hPSCs.
• To assess the implications of Cas9 toxicity for hPSC applications, including cell replacement therapies.

Main Methods:

• Utilized hPSC lines with stable Cas9 integration and transient delivery of Cas9-ribonucleoproteins (RNPs).
• Quantified insertion or deletion (indel) efficiency following CRISPR/Cas9-induced double-strand breaks (DSBs).
• Assessed the role of the P53/TP53 pathway in mediating cellular response to Cas9-induced DSBs.

Main Results:

• Achieved high average indel efficiencies exceeding 80% in hPSCs.
• Demonstrated that high-efficiency DSB induction by Cas9 is toxic to hPSCs, leading to cell death.
• Confirmed that Cas9 toxicity and reduced precise genome engineering efficiency in hPSCs are dependent on a wild-type P53/TP53 gene.
• Identified Cas9 toxicity as a significant obstacle to high-throughput CRISPR/Cas9 applications in hPSCs.

Conclusions:

• Cas9 toxicity, mediated by the P53/TP53 pathway, significantly impedes CRISPR/Cas9 genome engineering and screening in hPSCs.
• The findings necessitate caution and monitoring of P53/TP53 function in hPSCs intended for cell replacement therapies, especially those with acquired P53 mutations.