Programmed Manipulation of RNA Targets By Human Argonaute 2

Nucleic acid manipulation using programmable ribonucleoprotein complexes (RNPs) has enabled transformative research tools and led to new therapeutic strategies. RNA directly regulates diverse cellular processes,1 is a crucial mediator of protein synthesis, and offers advantages in therapeutic targeting and fundamental discovery complementary to those of DNA.2 ISC-3 and Cas-based4,5 scaffolds where the RNP is fused to an effector protein can alter RNA sequence, structure, and function. However, the non-human origins underlying these systems create challenges in therapeutic translation and the presence of non-native proteins can have unintended and little understood effects on cells.6-8 Systematic repurposing of human proteins, which have been optimized in the cellular environment by evolution, for expanded programmable functions could reveal new biological principles and bypass the limitations of foreign proteins. Here, we demonstrate that the catalytic engine of the RNA-interference (RNAi) pathway, human Argonaute 2 (AGO2),9,10 can be repurposed as a modular targeting domain, and when fused to a C-to-U deaminase, enable AGO-Led Targeted Editing of RNA (ALTER). Using guide RNAs which remodel target RNA structure for selective editing and reduced nuclease activity, we show that ALTER can act on a variety of target transcripts including endogenous mRNAs and lncRNAs, with activities comparable or exceeding those of Cas-based systems.11,12 Despite its human origin and role in RNAi, transcriptome-wide RNAseq revealed lower levels of off-target editing compared to Cas-based editing systems. These results demonstrate that AGO2 can be rationally redirected from RNAi to a broader spectrum of RNA manipulations, establishing that intact human proteins can be reconfigured for expanded molecular function.