## 2 Materials and Methods  
### Plasmid Construction  
We used enhanced green fluorescent protein (EGFP) W58X as the target gene for SDRE, which was prepared in a previous study [21]. Expression vectors for MS2 coat protein (MCP) and ADAR1 deaminase domain (ADAR1 DD) were also prepared in previous work [30].  
In the present study, MCP harbored an asparagine to lysine mutation at amino acid position 55 (N55K) because the mutation has higher binding affinity to MS2 RNA than to wild-type MCP [31]. The origin guide construct was prepared in a previous study [29]. The human _U6_ (_hU6_) promoter was substituted for the _CMV_ IE94 promoter in pCS2+ (Addgene, Watertown, MA, USA), and the guide RNA was placed downstream of the hU6 promoter. In the guide RNA sequence (5'-gaacatgaggatcacccatgtcggggcacggggcacggdcagtaacatgaggatcacccatgtctttttt-3'), the underlined parts are the MS2 stem-loop RNA, and bold indicates the antisense RNA region. Guide RNAs with different numbers of MS2 stem-loop RNAs on the 5'side of the antisense RNA and the 3'side of the antisense RNA were engineered based on the original parent construct with a stem-loop on either side of the antisense RNA (1-1 stem-loop in Figure 1).  
Figure 1: The performance of different numbers of MS2 stem-loop constructs. (**a**): Fluorescence micrographs of HEK293T obtained at 48 h after transfection. Images on the left panel are fluorescence images, the middle panel shows phase contrast images, and the right panel shows merged images. The far-left panel shows schematic diagrams of the guide RNAs. Sanger sequencing results. Black arrows indicate the target base for EGFP W58X. (**b**): Bar graph showing the editing efficiency (%) calculated from the sequencing results using the peak height ratio method. Results are mean \(\pm\) standard error of the mean (SEM n = 3). The editing level of EGFP W58X (negative control) was set at 0%. Asterisks indicate a statistically significant difference (\(p<-0.05\)). NS = not significant. (**c**): Bar graph showing the expression level of each type of guide RNA. The expression level of the 1-1 guide RNA (parent) construct was set as 1. Results are mean \(\pm\) SEM (n = 3).  
First, we prepared three types of constructs at downstream of the U6 promoter based on the original (1-1 stem-loop) guide RNA construct: 1-2 stem-loop, 2-1 stem-loop, and 2-2 stem-loop (Figure S1). The schematic diagram of their structure is shown in Figure 1. Second, to examine how each type of base pair in mismatches affects the editing efficiency, we used the 1-1 stem-loop guide RNA construct as a backbone, and replaced the C base in the mismatch position with A, U, or G. Then, all the six types of guide RNA construct were tested. Primers used for construction of these constructs are shown in Supplementary Table S1.  
Plasmids were transformed into Escherichia coli _DH5\(\alpha\)_ competent cells (TaKaRa Bio, Shiga, Japan). Positive clones were cultured in 5 mL Lysogeny broth (LB) medium with ampicillin at 37 \({}^{\circ}\)C for 18 h with shaking and extracted using NucleoBond Xtra Midi (MACHEREY-NAGEL, Germany). The concentration of isolated plasmid was determined using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).