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

Abstract

Abstract 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 mitotic entry with WEE1 inhibitors restores the toxicity of ATR inhibitors in CDC25A deficient cells. In addition, we have adapted the CRISPR-based SAM (Synergistic Activation Mediators) system to develop a doxycycline-inducible nuclease-dead Cas9 mESC line to induce gene expression at will. We generated mESC cell libraries by using a lentiviral library of 70000 sgRNAs designed against the proximal promoter of 20000 mouse genes to perform a gain-of-function screening with lethal doses of ATR inhibitor and results will be presented. With ATR inhibitors now entering the clinic, our work provides a better understanding of the mechanisms by which these compounds kill cells, and reveals genetic interactions that could be used for their rational use. Taking together, our loss-of-function and gain-of function cell libraries in mESCs represent an excellent platform to systematically identify genes involved in mediating resistance against chemotherapeutic agents. This abstract is also being presented as Poster A35. Citation Format: Sergio Ruiz, Cristina Mayor-Ruiz, Vanessa Lafarga, Matilde Murga, Maria Vega, Sagrario Ortega, Oscar Fernandez-Capetillo. Cas9/RNA-based forward genetic screenings in mouse embryonic stem cells uncovered the role of genes mediating resistance to ATR inhibitors [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR15.