Engineering of high-precision C-to-G base editors with expanded site selectivity and target compatibility

Base editors (BEs) are powerful tools for single nucleotide substitutions without requiring DNA double-stranded breaks or donor templates. The development of C-to-G base editors (CGBEs) represents a significant advancement by enabling base transversions, thus expanding the range of genetic modifications beyond traditional transitions and facilitating a broader spectrum of (therapeutic) applications. However, current CGBEs suffer from limitations in their editing range, mostly modifying position 6 relative to the distal end of the PAM, and their editing efficiency depends on the sequence context. In this study, by systematic exploration of deaminases to construct CGBEs, we have identified PmCDA1-based CGBEs that preferentially edit position 3. Furthermore, we report that truncations of the CDA1 C-terminus significantly enhance C-to-G editing efficiency. Our CDA1Δ-CGBEs not only exhibit high precision but also display remarkable compatibility with diverse substrate sequence contexts. We also show that they can substantially reduce, or even eliminate, genome-wide off-target editing. Importantly, we demonstrate that the strategy of using truncated CDA1 variants to improve C-to-G editing is effective not only in yeast but also in human and rice cells. These enhanced C-to-G base editing tools hold great promise for a wide range of applications in gene therapy, precision breeding, and fundamental research.