Development of an Efficient and Precise TadA-based C-to-G Base Editor Mi-zCGBE

C-to-G base editors (CGBEs) offer the potential to correct 11% of pathogenic single-nucleotide variants. While APOBEC-based CGBEs are highly efficient, they are limited by high indel rates and off-target activity. In contrast, TadA-based CGBEs offer greater precision but currently suffer from suboptimal editing efficiency. In this study, we first developed zCGBE, a TadA-based variant that achieves editing efficiency comparable to APOBEC-based editors while maintaining low Cas-independent off-target activity. To further reduce residual on-target indels, we engineered mi-zCGBE by fusing Brex27, a 36-amino-acid-long RAD51-recruiting peptide, to zCGBE. Mi-zCGBE preserves high C-to-G editing capacity and low Cas9-independent off-target activity while significantly reducing both on-target indels and guide-RNA-dependent off-target effects. We demonstrated the potential utility of mi-zCGBE by correcting the CFTR-N1303K mutation in Cystic Fibrosis patient-derived bronchial epithelial cells, achieving high rates of base correction and minimal indels or off-target edits, leading to restored CFTR protein expression and functional chloride channel activity. These results establish mi-zCGBE as an efficient and precise tool for gene editing research and future therapeutic applications.