###### 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.  
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Compared with DNA editing, RNA editing might be safer. RNA editing is a type of post-transcriptional modification of the gene-encoded sequence, which allows different forms of a protein to be produced from the same gene [15]. Mature messenger RNA (mRNA) consists only of exons; hence, intron editing does not occur, mRNAs are automatically degraded after translation is completed, and mutations are not passed on to offspring [16; 17; 18].  
Different methodologies for site-directed RNA editing (SDRE) have been developed to achieve gene editing at the RNA level. For adenosine (A)-to-inosine (I) RNA editing, SNAP-tag [19], \(\lambda N\) protein [20], MS2 system [21], and clustered regularly interspaced short palindromic repeats--associated protein 13 (Cas13) [22] systems have been reported. These technologies achieve SDRE in different ways, but they all follow the same principles.  
In mammalian cells, the conversion of A to I and cytosine (C) to uridine (U) occurs in both coding and noncoding sequences [23; 24; 25]. In human cells, apolipoprotein B mRNA editing enzyme (APOBEC1) was first found to replace C with U in APOB transcripts [26]. Meanwhile, A to I conversion, with I acting as guanosine (G) during translation, is mediated by members of the ADAR family [27; 28]. In recent years, ADAR2 has been used for performing C to U editing for certain mutation types [29].  
In previous studies, we optimized the MS2 system for SDRE [30]. We replaced the original cytomegalovirus (CMV) promoter with the human U6 promoter for guide expression and incorporated a flexible linker in the fusion protein. Next, we decreased the MS2 stem-loop RNA from six to two copies and placed the guide RNAs between the two stem-loop RNA. The optimization resulted in a significant improvement in editing efficiency [31].  
For the site-directed RNA editing system based on the MS2 system, previous studies did not compare the editing efficiency when the number of MS2 stem-loops and/or the mismatch base were changed. In the present study, we investigated the impact of these changes. We also explored the influence of different guide RNAs on guide RNA expression level and editing efficiency. The aim was to improve editing efficiency for practical applications.