A Targeted Genome-Wide Approach to Elucidate and Control Bacterial Adhesion to Physicochemically Diverse Biomaterials

Biofilm infections on indwelling biomedical devices are the leading cause of hospital infections around the world. The most common are catheter-associated infections caused by Escherichia coli and Staphylococcus aureus. Prevention and treatment of these infections typically rely on broad-spectrum antibiotics, yet these strategies often promote antibiotic resistance and are not fully effective. Elucidating the genetic elements responsible for bacterial adhesion to and biofilm formation on biomaterials could be used for the rational design of targeted antibiofouling agents that prevent infections on biomedical device surfaces. In this work, we aim to systematically map the genomes of E. coli and S. aureus to identify genetic targets that confer adhesion phenotypes and quantify their fitness using a multiplexed, high-throughput approach. In this approach, we design comprehensive genome-wide CRISPR interference (CRISPRi) gene repression libraries for E. coli and S. aureus and apply pooled selections on a platform of biomaterials (PEG and PDMS gels) that systematically vary in their physicochemical properties to uncover gene expression associated with bacterial attachment to various surfaces. We have developed and characterized CRISPRi tools in E. coli MG1655 using three CRISPRi systems to determine their design rules and appropriate expression levels to prevent cellular toxicity. Repression of genomic loci, including loci associated with biofilm formation, was quantified using superfolder GFP fusions. Using the determined design rules and custom Python scripts, we have designed gRNA libraries for each CRISPRi system (>40,000 designs) to target nearly every annotated gene in the MG1655 genome (95.9%–99.1%). For S. aureus, very few synthetic DNA parts have been characterized and reported in the literature. Here, we created and report what we believe to be the first characterized genetic part toolbox for S. aureus, including promoters, ribosome binding sites, terminators, and origins of replication. We then used the toolbox to rationally design CRISPRi tools for S. aureus.