Increasing the Editing Efficiency of the MS2-ADAR System for Site-Directed RNA Editing

Abstract

Site-directed RNA editing (SDRE) technologies have great potential in gene therapy. Our group has developed a strategy to redirect exogenous adenosine deaminases acting on RNA (ADARs) to specific sites by making editable structures using antisense RNA oligonucleotides. Improving the editing efficiency of the MS2-ADAR system is important in treating undesirable G-to-A point mutations. This work demonstrates an effective strategy to enhance the editing efficiency of this SDRE system. The strategy involves changing the number of MS2 stem-loops on both sides of the antisense RNA and the mismatch base on the antisense part. The enhanced green fluorescent protein (EGFP) with W58X mutation is used as the reporter gene. Subsequently, we adjusted the amount of plasmids for transfection to tune the expression level of the guide RNA, and finally, we observed the fluorescence signal after transfection. After equalizing number of MS2 stem-loops at both sides of the antisense RNA, high editing efficiency was achieved. In the same level of guide RNA expression, when the paired base position was the target uridine, the editing efficiency was higher than cytidine, adenosine, and guanosine.