Cas9 activates the p53 pathway and selects for p53-inactivating mutations
Cas9 activates the p53 pathway and selects for p53-inactivating mutations
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DOI：
10.1038/s41588-020-0623-4
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发表时间：
2020-05-18
期刊：
影响因子：
30.8
通讯作者：
Ben-David, Uri
中科院分区：
文献类型：
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作者：
Enache, Oana M.;Rendo, Veronica;Ben-David, Uri
Cas9通常被引入细胞系，以实现crispr -Cas9介导的基因组编辑。在这里，我们研究了Cas9表达本身的遗传和转录后果。165对人类癌细胞系及其表达Cas9衍生物的基因表达谱显示，引入Cas9后p53通路上调，特别是在野生型TP53 （TP53- wt）细胞系中。这在信使RNA和蛋白质水平上得到了证实。此外，在表达cas9的细胞系中观察到DNA修复水平升高。42对细胞系的遗传表征表明，Cas9的引入可导致p53失活突变的出现和扩增。在等基因tpp53 - wt和TP53-null （TP53(-/-)）细胞系的竞争实验中证实了这一点。最后，与p53突变细胞系相比，Cas9在p53 - wt中的活性较低，Cas9诱导的p53通路激活影响了细胞对遗传和化学扰动的敏感性。这些发现可能对正确使用crispr - cas9介导的基因组编辑具有广泛的意义。Cas9表达诱导DNA损伤，激活p53通路，可导致p53失活突变细胞的选择。Cas9在野生型TP53细胞系中的活性低于TP53突变细胞系。
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 itself. Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon introduction of Cas9, specifically in wild-type TP53 (TP53-WT) cell lines. This was confirmed at the messenger RNA 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 introduction of Cas9 can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53-WT and TP53-null (TP53(-/-)) 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.Cas9 expression induces DNA damage and activates the p53 pathway, and it can lead to the selection of cells with p53-inactivating mutations. Cas9 is less active in wild-type TP53 cell lines than in TP53-mutant cell lines.