Mycobacterial cell division arrest and smooth‐to‐rough envelope transition using CRISPRi ‐mediated genetic repression systems

The genetic basis underlying nontuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well‐established mycobacteria‐optimized dCas9 Sth1 ‐mediated gene knockdown system to develop a new subset of fluorescence‐based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use Mycobacterium smegmatis ( M. smeg ) and Mycobacterium abscessus ( M. abs ) as NTM model species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene silencing when targeting essential genes but also genes involved in smooth‐to‐rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.