PROJECT SUMMARY Alzheimer’s disease (AD) is a major personal and public health crisis in the United States and the incidence is increasing. Metabolic and inflammatory disorders contribute to AD risks by driving gene regulation and expression changes. Indeed, epigenetic changes during aging could reveal important therapeutic targets for preventing AD. The long-term goal of our work is to uncover the core cis-elements and gene regulatory networks (GRNs) that control AD risks. A challenge is that GRNs involve multiple cis-regulatory elements and genes across the genome. Currently, methods are lacking to functionally study combinations of cis-elements in vivo using mouse models, which is a barrier blocking us from identifying GRNs that can prevent or resolve AD pathology. The goal of this collaborative study between the Gregg and Gertz labs at the University of Utah is to develop an approach in mice to study the functions of combinations of cis-elements in vivo. The project will create a platform technology for functional studies of GRNs, facilitating the identification of conserved GRNs that can prevent or resolve AD pathology. The approach can be applied to many biological problems. In a recent Cell Systems paper, we (Gertz lab) devised a CRISPR- based technique that enables simultaneous epigenetic deactivation of multiple enhancers. The method is called Enhancer- interference (Enhancer-I) and can maintain stable silencing of up to 50 loci simultaneously. Enhancer-I was developed for in vitro studies and an Enhancer-I system for in vivo epigenome editing in mouse models does not yet exist. Therefore, we are collaborating with the Gregg lab to adapt Enhancer-I for in vivo work. The Gregg lab has extensive experience studying epigenetics and gene regulation in mice and recently published studies uncovering an atlas of conserved cis-elements and GRNs that are candidates for controlling processes involved in AD, including obesity, inflammation and neurodegeneration. Here, we will collaborate to create a mouse model of the Enhancer-I system for multiplexed in vivo epigenome editing. For proof-of-principle, we target cis-elements controlling Tau, beta-amyloid precursor protein (App), the Fat Mass & Obesity (FTO) locus and other important AD risk loci. Enhancer-I will empower researchers to study the functions of combinations of cis- elements and define functional GRNs controlling AD pathology in vivo. Our technology will help open new areas of study.