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

Abstract 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...

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Published in:Nature biotechnology 2024-07
Main Authors: Reautschnig, Philipp, Fruhner, Carolin, Wahn, Nicolai, Wiegand, Charlotte P., Kragness, Sabrina, Yung, John F., Hofacker, Daniel T., Fisk, Jenna, Eidelman, Michelle, Waffenschmidt, Nils, Feige, Maximilian, Pfeiffer, Laura S., Schulz, Annika E., Füll, Yvonne, Levanon, Erez Y., Mandel, Gail, Stafforst, Thorsten
Format: Article
Language:English
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Summary:Abstract 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.
ISSN:1087-0156
1546-1696
1546-1696
DOI:10.1038/s41587-024-02313-0