Fig. 1: Structure-based optimization of APOBEC3A enhances editing efficacy and reduces off-target effects of REWIRE-mediated RNA base editing.

a Schematic design of REWIREs with PUF variants. Top: Original CU-REWIRE 3.0 with PUF10, targeting 10-nt sequences. Bottom: Upgraded CU-REWIRE 4.0 with ePUF10. b Expression levels of CU-REWIREs in cultured cells (n = 3, technical replicates). Representative Western blot images using anti-Flag antibody, related to Supplementary Fig. 1b. c Crystal structure of Human APOBEC3A (PDB: 4XXO) highlighting putative dimerization interactions. Amino acids involved in dimer formation are shown in spherical forms. d Crystal structure of Human APOBEC3A (PDB: 5KEG) in complex with single-stranded DNA. Amino acids responsible for RNA recognition are labeled in spherical forms. Zn symbolizes the catalytic core. e Schematic illustration of APOBEC3A with its functional domains. f Schematic showing interaction between target EGFP mRNA and CU-REWIRE4.0 and 4.X with modified APOBEC3A variants. g Base editing efficiency of EGFP mRNA by CU-REWIRE4.X. The PUF binding site in EGFP is underlined in blue, with adjacent cytosines marked in red (top). Heatmap displays editing rates of cytosines near the on-target site C₄₅₉ for each CU-REWIRE and control (ePUF10 alone), with C₄₅₉ editing rate detailed on the right. Editing rates were measured by RNA-seq with triplicates (refer to methods). Values represent mean ± SEM. h Global editing rates of transcriptome-wide RNA base editing in samples treated with different CU-REWIRE4.X (related to data from g labels and cutoff as in Supplementary Fig. 1e). Orange rhombuses indicate the on-target site EGFP-C₄₅₉, and the mean number of off-target editing events is noted beside the dot plots. n represents the total number of edited cytosines detected. Values represent mean ± SD. i Correlation between on-target editing rates (Y-axis) and transcriptome-wide C-to-U off-target editing events (X-axis) for various CU-REWIRE4s.