Serial genome editing for multiple genes in slow-growing pathogens is extremely time consuming, and multiplexed genome editing can drastically expedite the genome editing progress. We assembled two sgRNA-expression cassettes into a single CTBEevolve plasmid to test the multiplexed editing capacity of CTBEevolve in the M. smegmatis mc 2 155 strain (Fig. ). As shown in Fig. , two genes (sigF and Ms6753) were simultaneously mutated in seven out of eight randomly picked colonies.