ABSTRACTThere is an unmet medical need for effective treatments againstMycobacterium abscessusinfections. Although advanced molecular genetic tools to validate drug targets and resistance ofM. abscessusexist, the practical design and construction of plasmids are relatively laborious and time-consuming. Thus, for this purpose, we used CRISPR interference (CRISPRi) combined with catalytically deactivated Cas9 to inhibit the gene expression of a predicted LysR-type transcriptional regulator gene,MAB_0055c, inM. abscessusand evaluated its contribution to the development of drug resistance. Our results showed that silencing the MAB_0055c gene lead to increased rifamycin susceptibility depending on the hydroquinone moiety. These results demonstrate that CRISPRi is an excellent approach for studying drug resistance inM. abscessus.IMPORTANCEIn this study, we utilized CRISPR interference (CRISPRi) to specifically target theMAB_0055cgene inM. abscessus, a bacterium that causes difficult-to-treat infections. The study found that silencing the gene lead to increased rifabutin and rifalazil susceptibility. This study is the first to establish a link between the predicted LysR-type transcriptional regulator gene and antibiotic resistance in mycobacteria. These findings underscore the potential of using CRISPRi as a tool for elucidating resistance mechanisms, essential drug targets, and drug mechanisms of action, which could pave the way for more effective treatments forM. abscessusinfections. The results of this study could have important implications for the development of new therapeutic options for this challenging-to-treat bacterial infection.