Meluzzi, D. | bioRxiv Science

bioRxiv2021

Robust RNA editing via recruitment of endogenous ADARs using circular guide RNAs

Akin to short-hairpin RNAs and antisense oligonucleotides which efficaciously recruit endogenous cellular machinery such as Argonaute and RNase H to enable targeted RNA knockdown, simple long antisense guide RNAs (1) can recruit endogenous adenosine deaminases acting on RNA (ADARs) to enable programmable A-to-I RNA editing, without requiring co-delivery of any exogenous proteins. This approach is highly specific, however the efficiency is typically lower than observed with enzyme overexpression. Conjecturing this was due in part to the short half-life and residence times of guide RNAs, here we engineer highly stable circular ADAR recruiting guide RNAs (cadRNAs), which can be delivered not only by genetically encoding on DNA vectors, but also via transfection of RNA molecules transcribed in vitro. Using these cadRNAs, we observed robust RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, vastly improved efficiency and durability of RNA editing, and high transcriptome-wide specificity. High transcript-level specificity was achieved by further engineering to reduce bystander editing. Additionally, in vivo delivery of cadRNAs via adeno-associated viruses (AAVs) enabled robust 38% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers, and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. Taken together, cadRNAs enable efficacious programmable RNA editing with application across diverse protein modulation and gene therapeutic settings.

bioengineering↗

bioRxiv2020

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

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.

synthetic biology↗