SummaryDual base editors (DBEs) enable simultaneous A‐to‐G and C‐to‐T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single‐strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE‐SpGn tools consisting of nine constructs using the high‐activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM‐flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG‐PAM). By analysing their editing performance on 48 targets in rice, we found that DBE‐SpGn constructs containing a single DBD and deaminases located at the N‐terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C‐terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high‐efficiency dual base editors (C‐A‐SpGn, C‐A‐D‐SpGn and A‐C‐D‐SpGn), named PhieDBEs (Plant high‐efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C‐A‐D‐SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C‐to‐T and A‐to‐G conversions as high as 81.0%. In summary, PhieDBEs (especially C‐A‐D‐SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.
