Book excerpt: RNA editing is defined as the insertion, deletion, or modification of a nucleotide that changes the information content of a sequence. Adenosine deaminases acting on RNA (ADARs) can deaminate an adenosine (A) in duplex RNA to inosine (I). Cellular machinery interprets inosine as guanosine, which can result in various consequences on RNA function. A-to-I editing can alter microRNA sequences, redirect splicing, and change secondary structure. More dramatically A-to-I editing can result in a recoding event, thereby changing the amino acid at a specific position. In recent years, there has been rapidly growing interest in engineering ADARs or directing endogenous ADARs to specific G-to-A mutations linked to various diseases. The contents of this dissertation details the progress we have made, with the help of various collaborations, to use ADARs for site- directed RNA editing. In chapter 1, I review various types of RNA editing with a great focus on adenosine deamination. I emphasize ADARs biological function, substrate specificity, and the roles ADARs have in various diseases. I further discuss the structural data that is known for ADAR2 and how this knowledge has led to a better understanding of using ADARs for site-directed RNA editing. In chapter 2, I discuss the previous approaches used for site-ivdirected RNA editing with ADAR and the challenge of overcoming off-target reactions. To overcome off-target reactions, I have designed an orthogonal editing system utilizing a bump-hole strategy to prevent off-target edits. I have shown that combining bulky ADAR mutants with a chemically modified guide RNA (gRNA) achieves site-selective editing with reduced off-target edits both in vitro and in cellular assays. In chapter 3, I focus on our collaboration with Prof. Gail Mandel's laboratory at Oregon Health and Science University to study a disease-causing mutation linked to Rett Syndrome. In this approach, we have focused on rationally designing chemically modified gRNAs that could potentially recruit endogenous wild type ADARs. Our rational design utilizes the crystallography of ADAR2 constructs bound to double stranded RNA (dsRNA) that were solved by our collaborators in Prof. Andrew Fisher's laboratory. In chapter 4, I deviate from using ADARs for site-directed RNA editing to elucidate the biological role of ADAR3. ADAR3 is catalytically inactive and is exclusively located in the brain. To further understand the role of ADAR3, five mutations were incorporated to engineer an active ADAR3 (ADAR3 M3). From here, we propose that ADAR3 not only acts as a negative regulator of ADAR1 and ADAR2, but also as a direct regulator in stabilizing specific transcripts. With an active ADAR3, future studies can be done to use ADAR3 M3 or another version of an active ADAR3 for site-directed RNA editing.