Application of the CRISPRi system to repress sepF expression in Mycobacterium smegmatis.

Despite technical advances in introducing genomic deletions and modulating gene expression, direct inactivation of essential genes in mycobacteria remains difficult. In this study, we described clustered regularly interspaced short palindromic repeat interference (CRISPRi) technology to repress the expression of sepF (MSMEG_4219) based on nuclease-deficient CRISPR-associated protein 9 (Cas9) and small guide RNA (sgRNA) specific to the target sequence in Mycobacterium smegmatis. Using this CRISPRi approach, we achieved the repression of sepF by up to 98% in M. smegmatis without off-target effects. The depleted Msm_sepF strains resulted in growth and morphology changes including elongated, filamentous and branched bacterial cells, but the levels of the interacting partners ftsZ and murG were not modified in M. smegmatis. The sepF gene was proven to be an essential gene in M. smegmatis. This study provided an improved and detailed technical procedure for the application of CRISPRi technology in mycobacteria, and this approach was demonstrated to be a simple and efficient tool for regulating the expression of essential genes in M. smegmatis.