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

ABSTRACT 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 . IMPORTANCE In 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.