A Multiplexed, Target-Based Phenotypic Screening Platform Using CRISPR Interference in Mycobacterium abscessus

The rise of difficult-to-treat Mycobacterium
abscessus 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 M.
abscessus, 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 M. abscessus space. The unexpected susceptibility to isoniazid, traditionally
considered to be ineffective in M. abscessus, suggested a complex interplay of several intrinsic resistance mechanisms.
While further complementary efforts will be needed to change the landscape
of therapeutic options for M. abscessus, 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.