Moreover, three different genomic sites (cysS, sigF and Ms6753) were targeted simultaneously by assembling three sgRNAs into the CTBEevolve plasmid, and all of the three targeted sites were successfully mutated in seven out of eight randomly picked colonies (Fig. ). Similarly, we assembled two or three sgRNA-expression cassettes into a single CGBEevolve plasmid and tested the multiplexed editing capacity of C-to-G conversion in the M. smegmatis mc 2 155 strain (Fig. and Fig. ). As shown in Fig. and Fig. , highly efficient multiplexed editing of C-to-G conversion was observed in both cases. Together, these results demonstrated that both CTBEevolve and CGBEevolve are amenable for multiplexed editing with high efficiencies in M. smegmatis.