CRISPR C-to-T base editors can convert CAA, CGA, CAG, and TGG codons to premature stop codons, and thereby are promising tools for gene inactivation . We examined the gene inactivation capacity of CTBEevolve by designing three different spacers targeting the essential L-leucine biosynthesis genes leuB and leuC. Efficient editing was observed for all the designed spacers (Fig. and F and Fig. ) and the isolated pure mutants were subjected to the phenotypical assay. Generating a premature stop codon in leuB or leuC renders the bacterium incapable of growth in the absence of L-leucine, confirming the inactivation of L-leucine biosynthesis (Fig. ). Moreover, similar strategy was successfully applied for inactivating ctpE by generating a premature stop codon with CTBEevolve. Consistent with previous studies , inactivation of ctpE by introducing the premature stop codon increased bacterium aggregation in the presence of EGTA (Fig. ). Together, these results confirmed that CTBEevolve is a powerful and reliable tool for gene inactivation in M. smegmatis.