Abstract PR15: Cas9/RNA-based forward genetic screenings in mouse embryonic stem cells uncovered the role of genes mediating resistance to ATR inhibitors

Mouse embryonic stem cells (mESCs) represent an excellent platform to study processes of developmental biology, model disease in vitro and in vivo or perform drug discoveries. In the last few years, the development of precise genome engineering tools based on the RNA-guided Cas9 nuclease from the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system, has enabled efficient and easy to engineer targeted genome modifications in mammalian cells. Using this technology, we generated a highly controlled doxycycline-inducible Cas9 mESC line, which harbor one single copy of Cas9 located in the type I collagen gene. Lentiviral delivery of small guide RNAs (sgRNAs) in doxycycline-treated mESCs demonstrated to be a very efficient manner to generate specific knockout mESCs lines. Therefore, we generated mESC libraries of knockout cells by using a lentiviral library of 80000 sgRNAs designed against 20000 mouse genes to perform forward genetic screenings. As a proof of concept, we investigated the existence of genes providing resistance to the absence of the ataxia-telangiectasia and Rad3 related (ATR) function by incubating the mESC libraries with lethal doses of ATR inhibitor. We here report the identification of CDC25A as a major determinant of sensitivity to ATR inhibition. CDC25A deficient cells resist high doses of ATR inhibitors, which we show is due to their failure to prematurely enter mitosis in response to the drugs. Forcing...
Source: Molecular Cancer Research - Category: Cancer & Oncology Authors: Tags: Replication Stress: Oral Presentations - Proffered Abstracts Source Type: research