Programmable C‐to‐U RNA editing using the human APOBEC 3A deaminase

Abstract

Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUES at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUES being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUES , can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.

ABCA4c.859-25A>G, a Frequent Palestinian Founder Mutation Affecting the Intron 7 Branchpoint, Is Associated With Early-Onset Stargardt Disease

“…Unfortunately, currently only A>G and C>U corrections can be obtained using engineered deaminase enzymes (such as ADAR and APOBEC), neither of which are viable options for our A>G variant. 71 – 73 …”

Site-directed RNA editing: recent advances and open challenges

“…A recently developed C to U editing tool is termed CURE (C to U RNA Editor) [ 61 ]. CURE fuses the APOBEC3A (A3A) cytidine deaminase to dCas13.…”

“…Thus, CURE has a high specificity towards C to U editing and shows significantly fewer target mutations than RESCUE. The specificity of CURE was further optimized by tightly controlling the intracellular localization by the addition of a nuclear localization signal which further reduced off target editing [ 61 ]. CURE-X uses CasRx instead of Cas13.…”

Immunity and Viral Infections: Modulating Antiviral Response via CRISPR–Cas Systems

“…Evolved ADAR2 protein exhibits dual A→I and C→U deaminase activity (RESCUE system) [ 174 ]. By replacing ADAR2 deaminase domain with APOBEC3A deaminase, it was possible to generate a C→U RNA-specific editase (CURE system) [ 175 ]. Additionally, truncated variants of Cas13 RNA editing tools (<1000 aa) that can be easily packaged into common AAV were engineered [ 176 ].…”