Combined CRISPR-Cas9 knockout and interference (CRISPRi) screening in M. tubeculosis.

High-throughput genetic screens are powerful methods to identify genes involved in a specific phenotype. Recently, CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR knockout (CRISPR-KO) screening, has emerged as the most powerful technology in genetic screening in eukaryotes. CRISPRi screening has been successfully used, but CRISPR-KO screening has not yet been applied for functional genomics studies in bacteria. Here, we constructed a genome-scale CRISPR-KO and CRISPRi library in Mycobacterium tuberculosis (Mtb). First, we used these two libraries to identify genes essential for Mtb viability. Next, we screened for genes whose loss is involved in resistance/susceptibility to bedaquiline, a therapeutic that inhibits mycobacterial F-ATP synthase and treats multidrug-resistant tuberculosis. The screens provided a valuable resource to investigate mechanisms of determining BDQ efficacy and identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. Our results established combined CRISPRi and CRISPR-KO screening as a powerful tool that provides rich and complementary information for high-throughput functional genomics to accelerate biological studies and drug developments in Mtb.