Precise in Vivo RNA Base Editing with a Wobble-enhanced Circular CLUSTER Guide RNA
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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'-UAN 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.
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An HIV-1 Reference Epitranscriptome.
Bosmeny M, Pater A, Zhang L, Sha B, Lyu Z, Larkai L bioRxiv. 2025; .
PMID: 39975020 PMC: 11838527. DOI: 10.1101/2025.01.30.635805.
Fry L, Major L, Salman A, McDermott L, Yang J, King A Commun Biol. 2025; 8(1):200.
PMID: 39922978 PMC: 11807095. DOI: 10.1038/s42003-025-07557-3.
A general strategy for engineering GU base pairs to facilitate RNA crystallization.
Ren Y, Lin X, Liao W, Peng X, Deng J, Zhang Z Nucleic Acids Res. 2024; 53(3).
PMID: 39721592 PMC: 11797044. DOI: 10.1093/nar/gkae1218.