Abstract
Recent studies have reported that genome editing by CRISPR–Cas9 induces a DNA damage response mediated by p53 in primary cells hampering their growth. This could lead to a selection of cells with pre-existing p53 mutations. In this study, employing an integrated computational and experimental framework, we systematically investigated the possibility of selection of additional cancer driver mutations during CRISPR-Cas9 gene editing. We first confirm the previous findings of the selection for pre-existing p53 mutations by CRISPR-Cas9. We next demonstrate that similar to p53, wildtype KRAS may also hamper the growth of Cas9-edited cells, potentially conferring a selective advantage to pre-existing KRAS-mutant cells. These selective effects are widespread, extending across cell-types and methods of CRISPR-Cas9 delivery and the strength of selection depends on the sgRNA sequence and the gene being edited. The selection for pre-existing p53 or KRAS mutations may confound CRISPR-Cas9 screens in cancer cells and more importantly, calls for monitoring patients undergoing CRISPR-Cas9-based editing for clinical therapeutics for pre-existing p53 and KRAS mutations.
Highlights
Recent studies have reported that genome editing by Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas[9] induces a DNA damage response mediated by p53 in primary cells hampering their growth
In the CRISPR-Cas[9] screen, we find many more genes (981) that are more essential in p53-WT vs. p53-mutant cell lines, compared to the genes that are more essential in p53-mutant cells (237 genes)
To determine whether additional cancer driver mutations may be selected for the following CRISPR-KO, we focused on a list of 61 cancer driver genes from Vogelstein et al.[26] that are mutated in at least 10 of the cell lines screened in the AVANA10 and Achilles[16] datasets
Summary
Recent studies have reported that genome editing by CRISPR–Cas[9] induces a DNA damage response mediated by p53 in primary cells hampering their growth. A study showed that exogenous expression of Cas[9] can activate this p53-mediated DNA damage response[15] While these studies indicate that CRISPR-Cas[9] genome editing techniques may select for p53-mutated cells[13,14,15], several outstanding questions remain unaddressed: First, since most of these p53 studies have involved only a small number of primary or transformed cells[13,14], it is unclear whether p53 selection can happen broadly across multiple different cell types including transformed cancer cells. We further identified the underlying pathways that are likely to mediate this selection
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