Enhancing RNA base editing on mammalian transcripts with small nuclear RNAs
Endogenous Uridine-rich small nuclear RNAs (U snRNAs) form RNA-protein complexes to process eukaryotic pre-mRNA into mRNA. Previous studies have demonstrated programmable U snRNA guide-targeted exon inclusion and exclusion. We investigated whether snRNAs can also enhance RNA base editing over state-of-the-art RNA-targeting technologies in human cells. Compared to adenosine deaminase acting on RNA (ADAR)-recruiting circular RNAs, we find that guided A>I snRNAs consistently increase adenosine-to-inosine editing for higher exon count genes, perturb substantially fewer off-target genes, and localize more persistently to the nucleus where ADAR is expressed. A>I snRNAs also more efficiently edit lncRNAs and pre-mRNA 3' splice sites to promote splicing changes. Finally, snRNA-H/ACA box snoRNA fusions (U>? snRNAs) increase targeted RNA pseudouridylation without DKC1 overexpression, facilitating improved CFTR rescue from nonsense-mediated mRNA decay in a Cystic fibrosis human bronchial epithelial cell model. Our results advance the endogenous protein-mediated RNA base editing toolbox and RNA-targeting technologies to treat genetic diseases. Overall design: RNA-seq of human HEK 293T cells following transfection with plasmids encoding pUC19 (empty vector), U7 promoter-driven U7smOPT snRNA (targeting DMD and RAB7A) in pUC19, and U6 promoter-driven cadRNA (targeting DMD and RAB7A) in pUC19