## 4 Discussion  
In this study, we showed that the number of stem-loops at both ends of the antisense RNA can affect the editing efficiency in SDRE (Figures 1 and 2). By adjusting the amount of construct used for transfection, the expression level of guide RNA was similar. Thus, the results could be compared reliably. Although changes in editing efficiency were difficult to detect by fluorescence observation, changes in editing efficiency could be seen more intuitively using Sanger sequencing. For the 2-2 stem-loop, although the expression level was lower than for the original parent construct, the editing efficiency was increased (Figure 2b,c). By contrast, for 1-2 stem-loop and 2-1 stem-loop, the editing efficiency was not altered after optimizing the amount of construct used for transfection (Figures 1b and 2b). These results suggest that when the number of stem-loops at both ends of the antisense RNA was the same, the MS2 SDRE system achieved higher editing efficiency. In previous studies, the molar ratio of guide RNA to target gene was found to affect editing efficiency, and this may have an unavoidable impact on the effectiveness of gene therapy. Although a high editing efficiency (up to 57%) was reported in one recent study using \(\lambda N\), the molar ratio of guide RNA expression plasmid (\(U6\) pENTR guide RNA vector) to target gene (CFTR Y122X) expression plasmid was 60:1, implying that the guide RNA was 60-fold more abundant than the target gene [25]. In another study on SDRE using Cas13, the molar ratio of guide RNA expression plasmid to target gene expression plasmid (RNA editing reporter) was 7.5:1 [22]. In our current study, the molar ratio of guide RNA expression plasmid to EGFP W58X was varied, the molar ratio of the 1-1 stem-loop in transfection was 59:1, compared with 36:1 for the 1-2 stem-loop, 44:1 for the 2-1 stem-loo, and 28:1 for the 2-2 stem-loop before optimization for transfection. After adjustment, the molar ratio was 39:1. Interestingly, after adjustment, the molar ratio of plasmid for transfection was similar, the 1-1 stem-loop and 2-2 stem-loop still achieved a similar level of editing efficiencies (Figure 2b), and the guide RNA expression level was similar for the 1-1 stem-loop and the 2-2 stem-loop (Figure 2c). It is difficult to explain this result, but it might be related to the interaction between the MS2 coat protein and the stem-loop of the MS2 RNA. It may also be due to the unequal number of stem-loops at both ends of the antisense RNA or the distance between the MS2 coat protein bound to the MS2 RNA and target sites will differ, thereby affecting the efficiency of RNA editing. Next, we verified whether different base mismatch pairings could affect editing efficiency. In a previous study, with a C mismatch, the editing efficiency was highest [33]. By contrast, our results showed that introducing a U base pair achieved the highest editing efficiency (Figures 3b and 4b). It is interesting because a highest editing efficiency may achieve when the bases are perfectly matched. It is difficult to explain this result, but it might be related to the structure of the ADAR1 catalytic domain. A perfectly matched double-stranded structure is more likely to bind to the ADAR1 catalytic domain and the target gene, thereby improving the catalytic efficiency. In the previous study, the E1008Q variant of ADAR1 had increased the editing efficiency of RNA editing on natural substrates and artificial substrates [22; 34]. For MS2-ADAR1 system, we guessed that the E1008Q variant of ADAR1 also affects editing efficiency. We performed the transfection of E1008Q variant of ADAR1 with 1-1 stem-loop guide RNA and reporter plasmid. However, the results showed that the editing efficiency of E1008Q variant of ADAR1 was the same as wild-type ADAR1. A possible explanation for this might be the distance between the mismatch base and the MS2 stem-loop RNA. The present study was designed to confirm the editing efficiency would be affected by the distance between the mismatch base and the stem-loop [22].