Cas9 activates the p53 pathway and selects for p53-inactivating mutations

Abstract

Cas9 is commonly introduced into cell lines to enable CRISPR/Cas9-mediated genome editing. Here we studied the genetic and transcriptional consequences of Cas9 expression per se . Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon Cas9 introduction, specifically in TP53 -WT cell lines. This was confirmed at the mRNA and protein levels. Moreover, elevated levels of DNA repair were observed in Cas9-expressing cell lines. Genetic characterization of 42 cell line pairs showed that Cas9 introduction can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53 -WT/ TP53 -null cell lines. Lastly, Cas9 was less active in TP53 -WT than in TP53 -mutant cell lines, and Cas9-induced p53 pathway activation affected cellular sensitivity to both genetic and chemical perturbations. These findings may have broad implications for the proper use of CRISPR/Cas9-mediated genome editing.

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells

“…Modulation of the p53 signaling pathway by Cas9 nucleases may be a more general feature. For example, the Cas9 protein of the bacterial pathogen Streptococcus pyogenes , SpyCas9, activates the p53 pathway in a wide variety of human cell lines [ 54 , 55 , 56 ].…”

Challenges and Strategies in Ascribing Functions to Long Noncoding RNAs

“…This damage can be repaired by nonhomologous end-joining (NHEJ), an error-prone repair method that induces indel mutations [ 40 ]. In the case of CNA, there are more sgRNA target sequences, which increases the chance of Cas9-mediated DNA cuts and double-strand break-induced antiproliferative effects [ 41 , 42 ], especially in p53 wild-type cells [ 43 , 44 , 45 ]. Another issue is that CNA of essential genes may protect them from complete knockout because Cas9-mediated genome editing is likely to be incomplete and because more copies of the target gene may increase resilience to knockout editing [ 39 ].…”

Therapeutic Editing of the TP53 Gene: Is CRISPR/Cas9 an Option?

“…Interestingly, Cas9 was reported to activate the p53 pathway and select for p53-inactivating mutations [ 56 ]. In this study, gene expression profiling of human cancer cell lines and their Cas9-expressing derivatives identified upregulation of the p53 pathway upon introduction of Cas9, particularly in TP53 WT cell lines.…”