Abstract
Background Cytidine base editors (CBEs) consist of a single-strand specific cytidine
deaminase fused to Cas9 nickase, enabling efficient C-to-T conversion across diverse
organisms. Enhancing editing range and efficiency of these tools is essential for
expanding their applications.
Results In this study, we report that fusing a double-stranded DNA-specific cytosine
deaminase DddAE1347A to CBEs significantly improves editing activity and broadens the
editing window in cell lines, embryos, tobacco, and cotton. Compared to BE4max, the
optimized DddAE1347A-BE4max exhibits up to a 93- fold increase in editing efficiency,
achieving up to 52% efficiency at C14 and C15 in cell lines. Further inv... More
Abstract
Background Cytidine base editors (CBEs) consist of a single-strand specific cytidine
deaminase fused to Cas9 nickase, enabling efficient C-to-T conversion across diverse
organisms. Enhancing editing range and efficiency of these tools is essential for
expanding their applications.
Results In this study, we report that fusing a double-stranded DNA-specific cytosine
deaminase DddAE1347A to CBEs significantly improves editing activity and broadens the
editing window in cell lines, embryos, tobacco, and cotton. Compared to BE4max, the
optimized DddAE1347A-BE4max exhibits up to a 93- fold increase in editing efficiency,
achieving up to 52% efficiency at C14 and C15 in cell lines. Further investigation
reveals that DddAE1347A is compatible with various Cas9 variants (SpCas9, SpaCas9, and
Nme2Cas9) and deaminase variants (rA1, A3G, and A3A). Additionally, we demonstrate
that cytosine deaminases with single-stranded DNA activity fail to enhance the CBE
system. In contrast, various DddA variants can improve CBE editing activity at PAMproximal cytosine positions, highlighting the modularity of fusion between DddAs and
CBEs.
Conclusions These findings suggest that the double-stranded DNA-specific cytosine
deaminase protein can act as an engineered fusion module in the CBE system, altering
the performance (window/efficiency) of CBEs.
