The identification of an expanding number of genetic susceptibility loci for Alzheimer's disease (AD) provides an opportunity for new mechanistic and therapeutic insights. The overall goal of this proposal is to bring together new genome engineering and stem cell technology to further our understanding of AD genetics and pathogenesis. The CRISPR-Cas9 system is a novel and facile platform for genome engineering that was described by us in the past year. We propose using the CRISPR-Cas system to generate a library of isogenic human IPSC lines with AD risk variants in each of the 10 susceptibility genes identified in multi-cohort GWAS studies. We have established 14 IPSC lines from dermal fibroblasts of late-onset AD (LOAD) and age-matched controls that have been differentiated to neurons and astrocytes. This led to the observation that IPSC-derived neural progenitors from LOAD patients differentiate to neurons prematurely, and differ in their transcriptional profiles when compared with controls. A similar phenotype was observed in AD vs control IPSC-derived neural progenitor lines from another laboratory. Informatic analysis implicated downregulation of a gene network controlled by the neural transcription factor REST/NRSF in AD neural progenitors. A similar change in this gene network was observed in vivo, in the brains of patients with mild cognitive impairment (MCI) and AD. Our preliminary studies suggest that REST also regulates genes involved in cell death pathways, AD pathology and inflammatory/immune responses. The generation of isogenic IPSC lines that differ only in the presence or absence of genetic variants associated with AD will enable us to explore the role of REST in a highly controlled manner, as well as investigate other pathogenic mechanisms involving A? metabolism, tau phosphorylation and neuronal stress responses. Furthermore, isogenic IPSCs will be differentiated into astrocytes and microglia to explore inflammatory phenotypes associated with risk alleles in the immune-related genes TREM- 2, CD33 and CR1. To elucidate the affected pathways, new high-sensitivity transcriptome sequencing and proteomic technology will be applied to IPSC-derived neurons and microglia with AD variants. Transcriptome data from isogenic cells will be integrated with RNA-seq analysis of the brain in subjects from the Religious Orders Study with the same genotypes. This approach will identify core regulatory mediators and pathways, with the goal of constructing a network model of AD risk variants. These studies will bring together two principal investigators and many collaborators with diverse but complementary areas of expertise in a multidisciplinary approach to understand genetic modifiers of AD.