Argonaute-mediated RNA editing selectively repairs point mutations

RNA editing enzymatically modifies RNA molecules post-transcriptionally, enabling precise sequence alterations. Advantages include reversibility and temporal control without genomic DNA changes, allowing dynamic regulation of gene expression while preserving original genetic information. In this study, we characterized McAgo derived from Monosporascus cannonballus, which functions as a programmable nuclease guided by 14-30 nt gRNAs, demonstrating robust RNA cleavage activity at physiological temperature. Furthermore, we delivered McAgo RNP (ribonucleoprotein) complexes into mammalian cells, achieving >90% RNA knockdown efficiency with minimal innate immune responses. A catalytically inactive mutant (dMcAgo) using a gRNA as short as 20 nt, conjugated to the hADAR2 deaminase domain (hADAR2dd E488Q), achieved up to 90% RNA editing efficiency in vitro. This study establishes, for the first time, the effective targeting of endogenous RNA by a heterologous Argonaute in mammalian cells, alongside its demonstrated utility for RNA editing-thereby expanding the functional repertoire of Argonaute proteins.