Application of <i>Ng</i>Ago-mediated genome editing in <i>Mycobacterium smegmatis</i>

Mycobacterium smegmatis is nonpathogenic and fast-growing and is usually used as a model species of Mycobacterium . Studying basic metabolic mechanisms is crucial for accelerating mycobacterial research. Although several tools for genome editing in Mycobacterium smegmatis MC (2) 155 ( M. smegmatis ) can be used, plasmids are difficult to construct, and the knockout efficiency is still low. Here, the Ng Ago system was utilized to edit the genome of the Gram-positive bacterium M. smegmatis , which has a high guanine-cytosine (GC) content. A shuttle plasmid containing the hsp60 promoter to drive Ng Ago expression was designed. PCR-mediated screening and qRT‒PCR confirmed that the glnR gene (KEGG: MSMEG_5784 ) and ltmA gene (KEGG: MSMEG_6479 ) were successfully knocked out by the Ng Ago-F system. The editing efficiency reached 80%, and the time requirement was reduced to 8 days. The optimized Ng Ago system establishes an efficient genome-editing platform for high-GC mycobacteria, advancing functional genomics research on M. smegmatis and potentially enabling precise interrogation of virulence mechanisms in pathogens, such as Mycobacterium tuberculosis .IMPORTANCEIn this work, we demonstrated that the Ng Ago system could be used to edit the genome of Mycobacterium smegmatis and has several advantages: easy plasmid construction, high editing efficiency, and short time requirements. These findings provide a powerful tool for elucidating the basic metabolic mechanisms of M. smegmatis and potentially those of other mycobacterial species.