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The introduction of CRISPR interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but technical challenges of the prototypical Cas9-based platform, for example in multigene regulation, remain. Here, we introduce an alternative CRSPRi platform that uses the minimal Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, and can regulate multiple genes simultaneously, providing a new tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb).

We screened a series of 4-anilinoquinolines and 4-anilinoquinazolines and identified novel inhibitors of Mycobacterium tuberculosis (Mtb). The focused 4-anilinoquinoline/quinazoline scaffold arrays yielded compounds with high potency and the identification of 6,7-dimethoxy-N-(4-((4-methylbenzyl)oxy)phenyl)quinolin-4-amine (34) with an MIC90 value of 0.63-1.25 μM. We also defined a series of key structural features, including the benzyloxy aniline and the 6,7-dimethoxy quinoline ring, that are important for Mtb inhibition. Importantly the compounds showed very limited toxicity and scope for further improvement by iterative medicinal chemistry.