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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 RESCUE‐S at the off‐targets transcriptome‐wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE‐S 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 RESCUE‐S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility. Synopsis: The current approach for programmable C‐to‐U RNA editing (RESCUE‐S) is limited by low editing efficiency at certain targets, the inability to edit nuclear RNAs, and non‐specific deamination of adenosines. Here, an alternative C‐to‐U RNA editing system—"CURE"—is introduced that complements RESCUE‐S in application. Fusion of the human cytidine‐specific deaminase APOBEC3A to dCas13 yields a distinct C>U RNA editor (CURE)The CURE system uses unconventional guide RNAs, which induce loops at target sites to imitate the natural substrate of APOBEC3ACURE and RESCUE‐S can complement each other in terms of editing motif preference, on‐target editing efficiency and off‐target effects. [ABSTRACT FROM AUTHOR]

Programmable A > I RNA editing is a valuable tool for basic research and medicine. A variety of editors have been created, but a genetically encoded editor that is both precise and efficient has not been described to date. The trade‐off between precision and efficiency is exemplified in the state of the art editor REPAIR, which comprises the ADAR2 deaminase domain fused to dCas13b. REPAIR is highly efficient, but also causes significant off‐target effects. Mutations that weaken the deaminase domain can minimize the undesirable effects, but this comes at the expense of on‐target editing efficiency. We have now overcome this dilemma by using a multipronged approach: We have chosen an alternative Cas protein (CasRx), inserted the deaminase domain into the middle of CasRx, and redirected the editor to the nucleus. The new editor created, dubbed REPAIRx, is precise yet highly efficient, outperforming various previous versions on both mRNA and nuclear RNA targets. Thus, REPAIRx markedly expands the RNA editing toolkit and illustrates a novel strategy for base editor optimization. Synopsis: Development of CRISPR/Cas9 programmable RNA base editors that exhibit both high efficiency and high specificity has proven challenging. A multipronged approach overcomes this limitation and allowed engineering of an optimized A > I RNA base editor, REPAIRx. Previously developed A > I editors based on ADAR2 deaminase fusion to dCas13b (REPAIRv1) cause substantial off‐target effects in the transcriptome.Off‐target effect‐reducing mutations in the ADAR2 deaminase domain (REPAIRv2) also significantly decrease on‐target editing.Fusion to an alternative targeting moiety (dCasRx), deaminase domain insertion in its middle, and nuclear localization of the fusion protein, yields a precise yet highly specific A > I editor.Nuclear localization of dCasRx allows editing of nuclear DNA in addition to cytoplasmic targets. [ABSTRACT FROM AUTHOR]

N 6-methyladenosine (m6A) is a common epitranscriptional mRNA modification in eukaryotes. Thirteen putative m6A readers, mostly annotated as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT) proteins, have been identified in Arabidopsis (Arabidopsis thaliana), but few have been characterized. Here, we show that the Arabidopsis m6A reader ECT1 modulates salicylic acid (SA)-mediated plant stress responses. ECT1 undergoes liquid-liquid phase separation in vitro, and its N-terminal prion-like domain is critical for forming in vivo cytosolic biomolecular condensates in response to SA or bacterial pathogens. Fluorescence-activated particle sorting coupled with quantitative PCR analyses unveiled that ECT1 sequesters SA-induced m6A modification-prone mRNAs through its conserved aromatic cage to facilitate their decay in cytosolic condensates, thereby dampening SA-mediated stress responses. Consistent with this finding, ECT1 overexpression promotes bacterial multiplication in plants. Collectively, our findings unequivocally link ECT1-associated cytosolic condensates to SA-dependent plant stress responses, advancing the current understanding of m6A readers and the SA signaling network., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)