Project Summary/Abstract RNA editing is a critical process for generating spatiotemporal transcriptomic diversity that is particularly important in the brain. A-to-I (adenosine to inosine, which is recognized as guanosine) editing is the most common form of RNA editing in metazoans and is catalyzed by a family of enzymes called adenosine deaminases acting on RNA (ADARs). A-to-I editing occurs co-transcriptionally when double-stranded RNA (dsRNA) is bound and edited by ADAR enzymes, which occurs at high frequency in the nervous system. Alteration of RNA editing levels is implicated in a number of neurological disorders. Loss of ADAR can lead to neurological phenotypes such as seizure, altered locomotion and circadian rhythm. Previous work studying mutations in ADAR demonstrates the importance of a few amino acids critical for proper editing activity, including a handful known to cause human diseases. However, we still lack a comprehensive understanding of ADAR protein function. We know even less about other trans regulators of RNA editing despite the evidence suggesting their existence. In this work, we aim to develop systematic approaches to deciphering the trans regulation of A-to-I RNA editing. First, we will identify functional mutants of ADAR1 and ADAR2 in human cells. Using a CRISPR-based technology we recently developed, we will perform saturation mutagenesis of ADAR1 and ADAR2 to introduce point mutations in human cells. We will identify functional ADAR mutants with decreased or increased editing activity and further characterize how these mutations affect ADAR editing activity in vivo. Second, we will identify novel regulators of RNA editing through biochemical and genetic screens. We will identify ADAR-interacting proteins in induced human neurons. We will also carry out a genome-wide CRISPR/Cas9 screen in induced human neurons to find candidates that alter editing levels. Top candidate genes are subject to secondary CRISPR/Cas9 screening in mouse primary neurons as well as double knockout in pairwise combinations to analyze their genetic interactions. Third, we will determine mechanistically how editing regulators alter the transcriptome-wide landscape of RNA editing. We will perturb the regulators to examine how they affect editing levels transcriptome-wide in human cells, mouse primary neurons, and Drosophila brains. We will test whether the regulators physically interact with ADAR1/2 or each other, and if and how they interact with ADAR RNA substrates. This work will provide an unprecedented understanding of trans regulation of A-to-I RNA editing in neurons, revealing novel mechanisms underlying this largely unexplored machinery.