Base editing is a powerful and precise genome-editing tool capable of introducing single-base substitutions with high resolution. Depending on the base-modification domains employed, base editors can mediate both transition and transversion base editing in mammalian cells. While deaminase-derived base editors have enabled efficient transition editing in the zebrafish genome, glycosidase-derived transversion editing remains a significant challenge. In this study, guided by structural insights into the Cas9 complex, we engineered a series of glycosidase guanine base editor (gGBE) constructs by inlaying the N-methylpurine DNA glycosylase (MPG) domain into Cas9 protein. Among these variants, we identified a high-performance gGBE in which MPG was inserted between residues G1246 and S1247 of the PI domain in Streptococcus pyogenes Cas9, which we named gGBE-PIGS. This variant improved editing efficiency by up to 3.5-fold in mammalian cells and 13-fold in zebrafish embryos, without compromising editing precision. Furthermore, we developed a gGBE-editing-responsive GFP reporter to monitor editing efficiency and enrich for gGBE-edited cells and embryos. Using a combination of the optimized gGBE-PIGS and the GFP reporter, we successfully generated zebrafish models carrying mutations homologous to human amyotrophic lateral sclerosis (ALS)-associated variants. Together, these findings highlight the robustness and versatility of the gGBE platform for precise base editing and underscore its promising potential for in vivo disease modeling and therapeutic genome engineering.
