Bacterial CRISPRi

Full Project Title

Developing a universal CRISPR-based interference system (CRISPRi) for bacterial gene regulation

Project Summary 

Deciphering functions of uncharacterised genes is a major challenge in microbiology. Loss-of-function experiments disrupting gene expression followed by phenotype analyses are a promising approach to address this challenge. The recently developed technology of clustered regularly interspaced short palindromic repeats interference (CRISPRi) allows targeted silencing of genes and operons, tuning of gene expression, and needs only small mutant libraries which is cost-saving. However, current CRISPRi workflows require expert knowledge, and the evaluation of phenotypic changes relies on mastering a multitude of bioinformatic tools. This project will evaluate recent CRISPRi method developments, optimise critical steps, and compile a user-friendly protocol for implementing CRISPRi in bacteria, including wet-lab and bioinformatics processes, to facilitate a wide uptake of this cutting-edge technology in microbiology across UQ.

Potential Outcomes

The main outcome of this project will be a widely applicable, user-friendly protocol for controlling bacterial gene regulation. The protocol will be a blueprint suitable for a diverse range of bacterial taxa, providing a considerable advantage for UQ researchers in terms of the scientific know-how created and cost savings by providing a ready to use workflow. This workflow will encompass a wet-lab protocol for an optimised, universal CRISPRi system that can be applied to a diverse range of target organisms, and a bioinformatics pipeline for CRISPRi phenotype evaluations covering the analysis of genomic, transcriptomic, and proteomic data.