A Multiplexed, Target-Based Phenotypic Screening Platform Using CRISPR Interference in <i>Mycobacterium abscessus</i>

The rise of difficult-to-treat <i>Mycobacterium abscessus</i> infections presents a growing clinical challenge due to the immense arsenal of intrinsic, inducible and acquired antibiotic resistance mechanisms that render many existing antibiotics ineffective against this pathogen. Moreover, the limited success in discovery of novel compounds that inhibit novel pathways underscores the need for innovative drug discovery strategies. Here, we report a strategic advancement in PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which is an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of bacterial mutants, each depleted of a different essential protein target, to identify whole-cell active compounds with high sensitivity. Applying this modified strategy to <i>M. abscessus</i>, in contrast to previously described versions of PROSPECT which utilized protein degradation or promoter replacement strategies for generating engineered hypomorphic strains, here we leveraged CRISPR interference (CRISPRi) to more efficiently generate mutants each depleted of a different essential gene involved in cell wall synthesis or located at the bacterial surface. We applied this platform to perform a pooled PROSPECT pilot screen of a library of 782 compounds using CRISPRi guides as mutant barcodes. We identified a range of active hits, including compounds targeting InhA, a well-known mycobacterial target but under-explored in the <i>M. abscessus</i> space. The unexpected susceptibility to isoniazid, traditionally considered to be ineffective in <i>M. abscessus</i>, suggested a complex interplay of several intrinsic resistance mechanisms. While further complementary efforts will be needed to change the landscape of therapeutic options for <i>M. abscessus</i>, we propose that PROSPECT with CRISPRi engineering provides an increasingly accessible, high-throughput target-based phenotypic screening platform and thus represents an important step toward accelerating early stage drug discovery.