p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells

Date: 
August 15, 2018
Place: 
HSW 1057

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1-3. 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 cells3-13. 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 induction3. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53gene 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 mutations14, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

Presenter: 
Hui Li
Publication Date: 
July 1, 2018
Presentation Paper: 
Time: 
12:00pm
Paper Author: 
Ihry RJ1, Worringer KA1, Salick MR1, Frias E2, Ho D1, Theriault K1, Kommineni S1, Chen J3, Sondey M4, Ye C5, Randhawa R1, Kulkarni T1, Yang Z2, McAllister G2, Russ C2, Reece-Hoyes J2, Forrester W2, Hoffman GR2, Dolmetsch R1, Kaykas A6.
Institution: 
1 Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
2 Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
3 Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
4 Abbvie, Cambridge, MA, USA.
5 Blueprint Medicine, Cambridge, MA, USA.
6 Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA, USA. [email protected]
Journal: 
Nat Med. 2018 Jul;24(7):939-946. doi: 10.1038/s41591-018-0050-6. Epub 2018 Jun 11.