A cytosine deaminase for programmable single-base RNA editing

Abstract

Adding to the RNA editing toolbox A previously developed RNA editing system called REPAIR can base edit A to I in RNA by fusing the adenine deaminase domain of ADAR2 with catalytically dead CRISPR-Cas13. Using directed evolution, Abudayyeh et al. demonstrated that the ADAR2 deaminase domain can be relaxed to accept other bases. This resulted in cytidine deamination activity, expanding the RNA editing toolbox for C-to-U conversion. This system, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), can edit on endogenous transcripts and enable modulation of posttranslational protein modification such as phosphorylation. Science, this issue p. 382 Programmable cytidine-to-uridine RNA editing with an evolved ADAR2 fused to CRISPR-Cas13 expands the RNA editing toolbox. Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive β-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.