Functional Genomics in Bacteria using CRISPR Interference

Jason M. Peters, Ph.D. Department of Microbiology and Immunology University of California, San Francisco

The ease of microbial genome sequencing has provided an unprecedented opportunity to uncover new essential genes and pathways that may serve as the next generation of antibiotic targets. To take advantage of this opportunity, we need organismindependent strategies capable of defining gene function for all genes, including essential genes and redundant genes in essential pathways. To address these issues, I established the following CRISPR interference (CRISPRi) approaches: 1) Chem-CRISPRi; combining partial knockdowns of essential genes with chemical probes to assign function to uncharacterized essential genes and identify the direct targets of antibiotics, 2) Double-CRISPRi; using multiplexed gene knockdowns to dissect genetic redundancy and identify new essential pathways and 3) Mobile-CRISPRi; CRISPRi systems that transfer to diverse, pathogenic bacteria and are compatible with Chem-CRISPRi and Double-CRISPRi. I will describe how I used my CRISPRi toolkit to identify the mode of action for a novel antibiotic, generate the first comprehensive map of essential gene function in bacteria, define essential pathways in the bacterial envelope, and to perturb essential gene functions in pathogenic bacteria that are associated with antibiotic resistance.