MAGPiE enables precise and versatile RNA manipulation via cleavage and ligation

<jats:p>RNA editing offers a promising alternative to permanent genome modification, yet most existing approaches remain largely confined to single-nucleotide substitutions. Here we present MAGPiE, a programmable RNA editing platform capable of diverse editing outcomes with limited site dependence. In MAGPiE1.0, target RNAs are site-specifically cleaved by a type III CRISPR complex and subsequently ligated by RtcB to ribozyme-generated RNA donors. Using a fluorescence reporter system, we established efficient receptor–donor ligation across multiple target sites and defined an optimal crRNA design. An optimized RtcB configuration, termed MAGPiE2.0, further enabled editing of endogenous mRNAs and noncoding RNAs with moderate efficiencies. To improve editing performance, we developed MAGPiE2.1–2.3 by introducing splint- or dCas13-mediated donor recruitment and by perturbing exoribonucleases. Receptor–donor base pairing that mimics native RtcB substrates increased editing efficiency by 2.6-fold in the MAGPiE3.0 framework and also provided the basis for an intron-targeting version. Beyond single-nucleotide substitutions, MAGPiE enables multi-nucleotide editing and programmable fragment replacement, including precise RNA-level knock-in for fluorescence tagging. Together, these results establish MAGPiE as a versatile platform that expands the scope of programmable RNA manipulation.</jats:p>