Combined CRISPR chemical-genetic screening of DLM, PMD and DETA/NO in Mycobacterium tuberculosis
The genetic determinants of Mtb sensitivity and resistance to DLM and PMD were analyzed by chemical-genetic profiling at the genome scale using a combined CRISPR screening platform. To repress target gene expression before treatment with DLM and PMD, the CRISPRi screening library was treated with anhydrotetracycline (ATc) for 3 days, after which both the CRISPRi and CRISPR-KO libraries were treated with ~1x MIC of DLM and PMD for 10 generations. The sgRNAs from the libraries were amplified and analyzed by next generation sequencing.