Engineered human Argonaute 2 for targeted C-to-U RNA base editing

Tools for manipulating RNA that rely on nonhuman proteins, such as ISC-based and Cas-based scaffolds, can lead to unintended and little-understood effects on human cells and present challenges in therapeutic translation. Replacing them with human proteins engineered for expanded, programmable functions could bypass the limitations of these foreign proteins. Here we demonstrate that human Argonaute 2 (AGO2) can be repurposed as a modular RNA targeting domain. AGO2 fused to a C-to-U deaminase enables AGO-led targeted editing of RNA (ALTER). Using guide RNAs that remodel target RNA structure for selective editing and reduced nuclease activity, we show that ALTER achieves, on average, 1.9-fold higher C-to-U editing on mRNA targets in HEK293T cells compared with Cas13 editors CURE and RESCUE. Transcriptome-wide RNA-seq revealed similar or lower levels of off-target editing compared to the same Cas13-based editors. Machine learning models predict that far fewer immunogenic peptides will result from ALTER and we show that ALTER is amenable to a validated workflow for artificial intelligence-directed deimmunization.