To evaluate the genome-wide off-target editing of the CTBEevolve system, two edited M. smegmatis colonies were randomly selected and subjected to whole-genome sequencing. No potential off-target editing sites that contain the identical sequences to the PAM proximal 1-8 nt of the protospacer were detected (Table ). These results demonstrated the high editing fidelity of the CTBEevolve system and are consistent with previous discoveries that St1Cas9 is a high-fidelity enzyme and its editing is highly sensitive to the mutations in the spacer sequences .