Journal Article

Precise in vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA

Hofacker, Daniel T., Feige, Maximilian, Reautschnig, Philipp, Fisk, Jenna, Levanon, Erez Y., Kragness, Sabrina, Füll, Yvonne, Pfeiffer, Laura S., Stafforst, Thorsten, Waffenschmidt, Nils, Yung, John F., Eidelman, Michelle, Mandel, Gail, Fruhner, Carolin, Schulz, Annika E., Wahn, Nicolai, Wiegand, Charlotte P.

2025

Overview

Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5′-U A N triplets, which dominate bystander editing, G•U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as G•A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo. Using G•U wobble base pairs at specific loci increases RNA base-editing precision and efficiency.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/45/500 / 639/166/985 / 692/308/2778 / 692/617/375/366 / Adenosine / Adenosine - genetics / Adenosine deaminase / Adenosine Deaminase - genetics / Adenosine Deaminase - metabolism / Agriculture / Animals / Base Pairing / Base pairs / Bioinformatics / Biomedical and Life Sciences / Biomedical Engineering/Biotechnology / Biomedicine / Biotechnology / Cell culture / Central nervous system / Clusters / Editing / Efficiency / Gene Editing - methods / HEK293 Cells / Humans / Life Sciences / MeCP2 protein / Methyl-CpG binding protein / Methyl-CpG-Binding Protein 2 - genetics / Mice / Rett syndrome / Ribonucleic acid / RNA / RNA editing / RNA Editing - genetics / RNA, Circular - genetics / RNA, Guide, CRISPR-Cas Systems - genetics / Uridine