Improvement of C-to-U RNA editing using an artificial MS2-APOBEC system

RNA cytidine deamination (C-to-U editing) has been achieved using the MS2-apolipoprotein B-editing catalytic polypeptide-like (APOBEC)1 editing system. Here, we fused the cytidine deaminase (CDA) enzymes APOBEC3A and APOBEC3G with the MS2 system and examined their RNA editing efficiencies in transfected HEK 293T cells. Given the single-stranded RNA preferences of APOBEC3A and APOBEC3G, we designed unconventional guide RNAs that induced a loop at the target sequence, allowing the target to form a single-stranded structure. Because APOBEC3A and APOBEC3G have different base preferences (5 '-TC and 5 '-CC, respectively), we introduced the D317W mutation into APOBEC3G to convert its base preference to that of APOBEC3A. Upon co-transfection with a guide RNA that induced the formation of a 14 nt loop on the target sequence, MS2-fused APOBEC3A and APOBEC3G showed high editing efficiency. While the D317W mutation of APOBEC3G led to a slight improvement in editing efficiency, the difference was not statistically significant. These findings indicate that APOBEC3A and APOBEC3G can induce C-to-U RNA editing when transfected with a loop guide RNA. Moreover, the editing efficiency of APOBEC3G can be enhanced by site-specific mutation to alter the base preference. Overall, our results demonstrate that the MS2 system can fuse and catalyze reactions with different enzymes, suggesting that it holds an even greater potential for RNA editing than is utilized currently. In this study, we utilized the MS2-APOBEC1 editing system to achieve RNA cytidine deamination (C-to-U editing) by fusing cytidine deaminase enzymes APOBEC3A and APOBEC3G with the MS2 system. We designed unconventional guide RNAs inducing a loop at the target sequence to facilitate single-stranded RNA preferences of APOBEC3A and APOBEC3G. The results demonstrate that APOBEC3A and APOBEC3G, when transfected with a loop guide RNA, can efficiently induce C-to-U RNA editing. Furthermore, the study suggests that the MS2 system has the potential to fuse and catalyze reactions with different enzymes, expanding its utility for RNA editing beyond current applications. image