To quantitatively assess the efficiency of targeted base editing, same amount of different CBE plasmids was electroporated into M. smegmatis. CBEdSt1Cas9 (dSt1Cas9 CBE) induced notable C-to-T conversions (from 4% to 15%) with undesired C-to-G conversions (from 18% to 70%) as the major editing products at all the test sites, whereas CBEdSpCas9 (dSpCas9 CBE) and CBEdLbCpf1 (dLbCas9 CBE) did not show detectable base editing activity at all the target regions (Fig. ). Moreover, consistent with previous studies , no notable cellular toxicity was observed for CBEdSt1Cas9 or CBEdLbCpf1, whereas high cellular toxicity existed with CBEdSpCas9 (Fig. ). Thereby, we chose CBEdSt1Cas9 as the starting system for further engineering. To cure the plasmid after base editing, we replaced the non-replicating L5 integrating backbone used in the CBEdSt1Cas9 system with the replicable pMF1 backbone, and found that the resulting editing plasmid did not show notable cellular toxicity and can be easily cured by culturing the bacterium in the absence of antibiotic (Fig. and, and Fig. and).