RT Journal Article
SR Electronic
T1 Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.09.08.288233
DO 10.1101/2020.09.08.288233
A1 Dhruva Katrekar
A1 Nathan Palmer
A1 Yichen Xiang
A1 Anushka Saha
A1 Dario Meluzzi
A1 Prashant Mali
YR 2020
UL http://biorxiv.org/content/early/2020/09/09/2020.09.08.288233.abstract
AB Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5’ guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2 protein engineering via three approaches: First, we performed a novel deep mutational scan of the deaminase domain that enabled direct coupling of variants to corresponding RNA editing activity. Experimentally measuring the impact of every amino acid substitution across 261 residues, i.e. ~5000 variants, on RNA editing, revealed intrinsic domain properties, and also several mutations that greatly enhanced RNA editing. Second, we performed a domain-wide mutagenesis screen to identify variants that increased activity at 5’-GA-3’ motifs, and discovered novel mutants that enabled robust RNA editing. Third, we engineered the domain at the fragment level to create split deaminases. Notably, compared to full-length deaminase overexpression, split-deaminases resulted in >1000 fold more specific RNA editing. Taken together, we anticipate this comprehensive deaminase engineering will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.Competing Interest StatementD.K. and P.M. have filed patents based on this work. P.M. is a scientific co-founder of Shape Therapeutics, Seven Therapeutics, Navega Therapeutics, Boundless Biosciences, and Engine Biosciences. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.