An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing.

ABSTRACT: Custom RNA base editing exploiting the human Adenosine Deaminase Acting on RNA (ADAR) enzyme may enable therapeutic gene editing without DNA damage or use of foreign proteins. ADAR's adenosine-to-inosine (effectively A-to-G) deamination activity can be targeted to transcripts using an antisense guide RNA (gRNA), but efficacy is challenged by limits of in vivo delivery. Embedding gRNAs into a U7 small nuclear RNA (snRNA) framework greatly enhances RNA editing with endogenous ADAR, and a 750-plex single-cell mutagenesis screen further improved the framework. An optimized scaffold with a stronger synthetic U7 promoter enables 76% RNA editing in vitro from a single DNA construct per cell, and 75% editing in a Hurler syndrome mouse brain after one systemic AAV injection, surpassing circular gRNA approaches. The technology also improves published DMD exon-skipping designs 25-fold in differentiated myoblasts. Our engineered U7 framework represents a universal scaffold for ADAR-based RNA editing and other antisense RNA therapies.

SUBMITTER: Byrne SM 

PROVIDER: S-EPMC12106830 | biostudies-literature | 2025 May

REPOSITORIES: biostudies-literature