Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

Abstract

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.

RNA binding by ADAR3 inhibits adenosine-to-inosine editing and promotes expression of immune response protein MAVS

“…Based on that study and to reduce the number of changes to wildtype ADAR3, two mutations, E527K and Q549R, were introduced into the ADAR3 lentiviral vector. The ADAR3 E527 residue is analogous to E488 in ADAR2, which, when mutated to glutamine (Q) or lysine (K), results in hyperediting activity ( 59 ). For ADAR3, E527K is one of the highly recurrent missense mutations identified in the ADAR3 deaminase domain in the Catalogue of Somatic Mutations in Cancer (COSMIC) database ( 60 ).…”

Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold

“…We next tested if CREST architecture can be used to support split ADAR2-DD to reduce dCas13-independent off-target RNA editing events. We split ADAR2-DD into two parts, the N-terminal and C-terminal fragments as reported (37), and fused them with ddCas13b or PUFc. We speculated that CREST is able to reconstitute ADAR2-DD catalytical activity at the target locus in two scenarios.…”

“…An additional application of our CREST system is its utility in potentially reducing dCas13-indpendent off-target effects encountered when full-length effectors are utilized (17). Restoring enzymatic activity of split ADAR at desired locus was shown to be an effective strategy to reduce off-target effect on both RNA and DNA base editing (37, 43). We demonstrated the reconstitution of ADAR2-DD from two split halves using CREST architecture(37).…”

“…Restoring enzymatic activity of split ADAR at desired locus was shown to be an effective strategy to reduce off-target effect on both RNA and DNA base editing (37, 43). We demonstrated the reconstitution of ADAR2-DD from two split halves using CREST architecture(37). Strikingly, compared with the full length PUFc-ADAR2-DD, our CREST system can reconstitute near 80% of catalytical activity of split ADAR2-DD and decrease about 96% of off-target events.…”