Abstract Phenotypes are often not only based on the effect of a single gene, but are rather influenced by multiple genetic (also termed epistatic) interactions. Prominent examples are synthetic lethal interactions, where the individual loss-of-function of two genes does not lead to phenotypic alterations, whereas their simultaneous perturbation results in reduced fitness or even cell death. Previously, we have established large-scale genetic interaction mapping experiments in Drosophila cells that discovered a high-number of functional interactions of conserved factors. Here, we aim to create a platform to investigate epistatic interactions of genes encoding chromatin remodeling and signaling factors in human cells. As part of the transcriptional regulatory machinery, chromatin remodeling enzymes buffer various genetic perturbations via adaptations within the transcriptional program, e.g. via repression of mutant or activation of functional alleles. Furthermore, chromatin remodeling factors link epigenetic modifications to corresponding signaling pathway outputs. While some of these enzymes have been extensively studied, many remain largely uncharacterized. As epigenetic dysregulation constitutes a characteristic feature of many cancers, chromatin remodeling proteins are promising targets in anticancer therapies. To map interaction networks of chromatin and signaling factors, we use pooled genome-scale CRISPR/Cas9 genetic interaction screens. To this end, CRISPR/Cas9 mapping will be performed in a human cell line to reveal unknown functions and interactions of chromatin remodeling enzymes. We will present our screening platform, including custom library design and analysis tools to identify screening hits that emerge to be promising for synthetic lethality targeting in anticancer therapy. Citation Format: Luisa Henkel, Florian Heigwer, Jan Winter, Benedikt Rauscher, Michael Boutros. Epistatic mapping of signaling and chromatin regulators [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B16.