Chemogenomic approaches to antibiotic drug discovery

This is an interdisciplinary program involving the Hu Lab and and the Davis Research Group.

We are building bacterial knockdown libraries as genetic tools to predict antibiotic activity in new compounds and to determine their mechanism of action. Our collections contain knockdown mutants in essential genes, which usually code for targets of antibiotics. The overarching hypothesis of our research is that  chemogenetic profiles of essential gene knockdowns will better predict antibiotic activity and mode of action (MOA) of novel compounds, accelerating antibiotic discovery. Our libraries are valuable tools for machine learning based prediction of antibiotic activity and mode of action of any active antimicrobial compound.

To increase the throughput of our approaches, we are constructing transposon based knockdown libraries where mutants can be tracked in pooled screens by PCR amplification of a DNA barcode inserted within the transposon. This method is called  Bar-seq.

We are also developing non-redundant, ordered, knockdown libraries with CRISPR interference (CRISPRi).

In our system, CRISPRi silencing of specific gene(s) requires the rhamnose-induced expression of a dead Cas9 endonuclease (dCas9), and a target-specific guide RNA (sgRNA).

Knockdown mutants have different susceptibility to molecules with antibiotic activity, which can signal drug-target interactions. CRISPRi mutants of gyrA and gyrB are specifically hypersusceptible to the gyrB-binding antibiotic novobiocin. CRISPRi for Burkholderia is available through Addgene.