Programmable RNA base editing with a single engineered protein

Programmable base editing of RNA enables rewriting the genetic codes on specific sites. Current tools for specific RNA editing dependent on the assembly or recruitment of the guide RNA into an RNA/protein complex, which may cause delivery barrier and low editing efficiency. Here we report a new set of tools, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. The REWIRE system contains a human-originated programmable RNA-binding domain (PUF domain) to specifically recognize target sequence and different deaminase domains to achieve A-to-I or C-to-U editing. By utilizing this system, we have achieved editing efficiencies up to 80% in A-to-I editing and 65% in C-to-U editing, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. We applied the REWIREs to correct disease-associated mutations and modify mitochondrial RNAs, and further optimized the REWIREs to improve the editing efficiency and minimize off-target effects. As a single-component base editing system originated from human proteins, REWIRE presents a precise and efficient RNA-editing platform with broad applicability in basic research and gene therapy. To assess the editing efficiency on target reporters, cells were plated in a 6-well plates (at approximately 70% confluency) and transfected 12 hours later with 2μg REWIRE expression vector and 200ng reporter plasmid, and were harvested 48 hours after transfection for further analyses. For the endogenous transcripts, HEK 293T cells were seeded on a 6-well plate (at approximately 70% confluency) and transfected 12 hours later with 2μg REWIRE expression plasmids using Lipofectamine 3000, then harvested cells 48 hours later. All transfections were performed with Lipofectamine 3000 (Thermo Fisher Scientific) in 6-well plates coated with poly-D-lysine (BD BioCoat).