Rationally Designed Pooled CRISPRi-Seq Uncovers an Inhibitor of Bacterial Peptidyl-tRNA Hydrolase

Area of Science:

• Microbiology
• Genetics
• Drug Discovery

Background:

• Pooled knockdown libraries are crucial for identifying antibacterial compound mechanisms.
• Uneven mutant proliferation in pooled libraries can lead to loss of essential gene targets.
• CRISPR interference (CRISPRi) offers a method to create targeted gene knockdown libraries.

Purpose of the Study:

• To develop a robust pooled knockdown library for comprehensive genomic coverage of essential genes in antibiotic-resistant bacteria.
• To overcome limitations of traditional pooled libraries in maintaining mutant representation.
• To enable efficient chemical-genetic profiling for novel antimicrobial target identification.

Main Methods:

• Construction of CIMPLE (CRISPRi-mediated pooled library of essential genes) in *Burkholderia cenocepacia*.
• Analysis of growth parameters from arrayed CRISPRi library to model and adjust mutant abundance for pooled growth.
• Application of CIMPLE for chemical-genetic profiling and CRISPRi-Seq to identify antimicrobial targets.

Main Results:

• CIMPLE achieved near-complete genomic coverage of essential gene knockdowns during pooled growth.
• Benchmarking with known antibacterials validated CIMPLE's efficacy in chemical-genetic profiling.
• Identification of peptidyl-tRNA hydrolase (Pth) as the target of a novel antibacterial compound.

Conclusions:

• CIMPLE effectively integrates arrayed and pooled CRISPRi library advantages for enhanced antibiotic target discovery.
• The developed library enables uncovering previously inaccessible antibacterial targets.
• This approach significantly advances the identification of mechanisms of action for novel antimicrobial agents.