Abstract The Hippo pathway is a signaling pathway that is crucial to regulate tissue homeostasis and organ growth. The core of the Hippo pathway consists of the MST1/2 and LATS1/2 kinase cascade that regulates the activity of the transcriptional coactivators YAP/TAZ by phosphorylation. Unphosphorylated YAP/TAZ display nuclear localization and interact with TEAD proteins to regulate gene expression. A tight regulation is crucial, since deregulated YAP/TAZ can lead to several organismal defects including cancer development. To ensure robustness of the signaling pathway to perturbations, many Hippo components are organized in a redundant manner, e.g., redundancy of LATS kinases. This feature aggravates the identification of new Hippo signaling components using loss-of-function screens since a knockout of one factor could potentially be compensated for by its redundant counterpart. To discover novel potential regulators of YAP, we performed a genome-scale gain-of-function CRISPR/Cas9 screening in MCF10A cells using the synergistic activation mediator (SAM) system. SAM is an adaptation of CRISPR/Cas9 to overexpress endogenous genes by targeting the proximal promoter with sgRNAs rather than knocking out target genes. Using the nuclear localization of YAP as a measure of YAP activity, we could identify several genes as positive regulators of YAP. One of the best screening hits is ISOC1 (Isochorismatase domain-containing protein 1). We validated the screening by using single sgRNAs to induce the endogenous ISOC1 locus by SAM. The expression of YAP-target genes as ANKRD1, CTGF, and IGFBP3 was increased upon ISOC1 overexpression. Main goals in the future will be to understand the biochemical mechanism by which ISOC1 modulates YAP activity and the biologic role of ISOC1. Since ISOC1 is reported to localize to peroxisomes and is induced upon dietary restriction in mice, we hypothesize that ISOC1 acts as a central hub to connect peroxisomal metabolism and YAP/TAZ activity. Citation Format: Paul Cramer, Björn von Eyss. Identification of YAP modulators using genome-wide gain-of-function screening [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B24.