Journal Article

p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells

Yang, Zinger, Worringer, Kathleen A., Salick, Max R., Russ, Carsten, Hoffman, Gregory R., Kulkarni, Tripti, Frias, Elizabeth, Ye, Chaoyang, Dolmetsch, Ricardo, Forrester, William, Chen, Julie, Ihry, Robert J., Ho, Daniel, Theriault, Kraig, Kommineni, Sravya, McAllister, Gregory, Sondey, Marie, Randhawa, Ranjit, Kaykas, Ajamete, Reece-Hoyes, John

2018

Overview

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells 1 – 3 . Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells 3 – 13 . Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction 3 . The toxic response to DSBs was P53/TP53 -dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations 14 , cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function. CRISPR–Cas9-induced DNA damage triggers p53 to limit the efficiency of gene editing in human pluripotent cells.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/136/2444 / 631/1647/1511 / 631/1647/1513/1967 / 631/532 / Biomedical and Life Sciences / Biomedicine / Cancer Research / Clonal deletion / CRISPR / CRISPR-Associated Protein 9 - metabolism / CRISPR-Cas Systems - genetics / Cyclin-Dependent Kinase Inhibitor p21 - genetics / Cyclin-Dependent Kinase Inhibitor p21 - metabolism / DNA / DNA Breaks, Double-Stranded / DNA damage / DNA sequencing / Efficiency / Engineering / Engineers / fas Receptor - genetics / fas Receptor - metabolism / Gene Deletion / Gene expression / Genes / Genetic Engineering / Genome editing / Genomes / Genomics / Humans / Infectious Diseases / Insertion / Letter / Metabolic Diseases / Molecular Medicine / Neurosciences / p53 Protein / Pluripotency / Pluripotent Stem Cells - metabolism / Polymerase chain reaction / Ribonucleoproteins / RNA, Guide, CRISPR-Cas Systems - metabolism / RNA, Messenger - genetics / RNA, Messenger - metabolism / Stem cell transplantation / Stem cells / Toxicity / Transcription, Genetic / Transfection / Tumor cell lines / Tumor proteins / Tumor Suppressor Protein p53 - metabolism / Tumors