Efficient glycosylase-mediated base editing with minimal off-target effects in mammalian embryos

Developing glycosylase-based base editors (gBEs) to broaden the editing scope is highly desirable for biomedical research and agricultural applications. However, the off-target effects and applicability of gBEs need further investigation. We employ GOTI to detect rare DNA off-target events in mouse embryos injected with N-methylpurine glycosylase-based AYBE and gGBE. Transcriptome-wide RNA analysis reveals that TadA8e-V106W, derived from AYBE, induces low-frequency RNA off-target editing. Both base editors efficiently induce A/G-to-Y editing in mouse and sheep embryos, and in newborn lambs. The robust efficiency and specificity of AYBE and gGBE underscore their potential for clinical applications and genetic improvement in livestock.