A cytosine deaminase for programmable single-base RNA editing

Abstract

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.

Figure 1:. Evolution of an ADAR2 deaminase domain for cytidine deamination

A. Schematic of RNA targeting of the catalytic residue mutant (C82R) of Gaussia luciferase reporter transcript. B. Heatmap depicting the percent editing levels of RESCUEr0-r16 on cytidines flanked by varying bases on the Gluc transcript. More favorable editing motifs are shown at the top, while less favorable motifs (5Ć) are shown at the bottom. C. Editing activity of RESCUE on all possible 16 cytidine flanking bases motifs on the Gluc transcript with U-flip or C-flip guides. D. Activity comparison between RESCUE, ADAR2dd without Cas13, full-length ADAR2 without Cas13, or no protein. E. Editing efficiency of RESCUE on a panel of endogenous genes covering multiple motifs. The best guide for each site is shown with the entire panel of guides displayed in fig. S19.

Figure 2:. Phenotypic outcomes of RESCUE on cell growth and signaling

A. Schematic of β-catenin domains and RESCUE targeting guide. B. Schematic of β-catenin activation and cell growth via RESCUE editing. C. Percent editing by RESCUE at relevant positions in the CTNNB1 transcript. D. Activation of Wnt/β-catenin signaling by RNA editing as measured by β-catenin-driven (TCF/LEF) luciferase expression. E. Representative microscopy images of RESCUE CTNNB1 targeting and non-targeting guides in HEK293FT cells. F. Quantitation of cellular growth due to activation of CTNNB1 signaling by RNA editing in HEK293FT cells.

Figure 3:. RESCUE and REPAIR multiplexing and specificity enhancement via guide engineering

A. Schematic of multiplexed C to U and A to I editing with pre-crRNA guide arrays B. Simultaneous C to U and A to I editing on CTNNB1 transcripts C. Schematic of rational engineering with guanine base flips to prevent off-target activity at neighboring adenosine sites. D. Percent editing at on-target C and off-target A sites for Gaussia luciferase (left) and KRAS (right) using rational introduction of disfavored base flips.

Figure 4:. Transcriptome-wide specificity of RESCUE

A. On-target C to U editing and summary of C to U and A to I transcriptome-wide off-targets for RESCUE compared to REPAIR. B. Manhattan plots of RESCUE A to I (left) and C to U (right) off-targets. The on-target C to U edit is highlighted in orange. C. Schematic of ADAR2dd interactions with RNA. Residues mutated for improving specificity are highlighted in red. D. Luciferase values for C to U activity with a targeting guide (y-axis) and A to I activity with a non-targeting guide (x-axis) shown for RESCUE and 95 RESCUE mutants. RESCUE is highlighted in red and mutants with better specificity in blue. The T375G mutant (REPAIRv2) is shown in orange. E. On-target C to U editing and summary of C to U and A to I transcriptome-wide off targets of RESCUE, REPAIR, and top specificity mutants. F. Manhattan plot of RESCUE-S (+S375A) A to I (left) and C to U (right) off-targets. The on-target C to U edit is highlighted in orange. G. Representative RNA sequencing reads surrounding the on-target Gluc editing site (blue triangle) for RESCUE (left) and RESCUE-S (right). A to I edits are highlighted in red; C to U (T) edits are highlighted in blue; sequencing errors are highlighted in yellow.