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

Area of Science:

• Microbiology
• Drug Discovery
• Genetics

Background:

• Mycobacterium abscessus infections are difficult to treat due to extensive antibiotic resistance.
• Limited success in discovering novel antimicrobial compounds necessitates innovative drug discovery strategies.

Purpose of the Study:

• To advance the PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) antimicrobial discovery strategy for Mycobacterium abscessus.
• To leverage CRISPR interference (CRISPRi) for efficient generation of essential gene-depleted mutants.

Main Methods:

• Modified PROSPECT strategy using CRISPRi to create pooled essential gene-depleted mutants in M. abscessus.
• Screened a library of 782 compounds using CRISPRi guides as mutant barcodes.
• Identified chemical-genetic interactions to find whole-cell active compounds.

Main Results:

• Identified active compounds, including those targeting InhA, a key mycobacterial enzyme.
• Discovered unexpected susceptibility to isoniazid, suggesting complex resistance mechanisms.
• Demonstrated the utility of PROSPECT with CRISPRi for M. abscessus drug discovery.

Conclusions:

• PROSPECT combined with CRISPRi engineering offers an accessible, high-throughput screening platform.
• This approach accelerates early-stage drug discovery for challenging M. abscessus infections.
• Further research is needed to develop new therapeutic options for M. abscessus.