To systemically characterize the PAM preference of CTBEevolve in vivo, we assembled 48 spacers targeting 48 different endogenous genomic sites with 3 spacers for each 5'-NNNNAA-3' PAM. We collected all the colonies after each transformation and subjected the PCR products of the target sites for sequencing to evaluate the editing efficiencies. Consistent with the in vitro DNA cleavage assay, CTBEevolve has an expanded PAM preference with the 5'-NNNNAA-3' PAM specificity (Fig. ). We also noticed that when targeting the sites with less-active PAMs, such as 5'-NNTAAA-3', 5'-NNTCAA-3', 5'-NNTGAA-3', and 5'-NNTTAA-3', the spacer sequence content can significantly affect the editing efficiency of CTBEevolve (Fig. ). Moreover, we analyzed the editing window of CTBEevolve, revealing that CTBEevolve preferred to edit Cs within the window from positions 4 to 12 (Fig. ). When the Cs were located outside the window from positions 4 to 12, but inside the window from 2 to 15, the Cs could sometimes still be edited with high efficiencies (Fig. ).