CRISPR interference knockdown screen identifies novel proteins involved in formation of structured macrocolonies in Staphylococcus aureus.

Staphylococcus aureus biofilms play important roles during infection. The main components of these biofilms are well studied; however, we lack the full understanding of factors and genes involved in regulation of biofilm formation. To screen for essential and non-essential biofilm regulatory genes in S. aureus, we have created a pooled inducible CRISPR interference library. The pooled library is designed to allow knockdown of every transcriptional unit in the S. aureus genome, thus targeting both essential and non-essential genes. We used our library in S. aureus Newman, a strain which forms structured macrocolonies on agar plates. We performed an unbiased screen of 1500 macrocolonies and found 10 macrocolonies with stably altered structures. The genotypes of these macrocolonies were determined by sequencing the single guide RNAs of the CRISPR interference system. As a proof of the validity of the approach, we identified several genes previously reported to be implicated in biofilm and macrocolony formation, including ica-genes, and metabolic genes of the TCA-cycle and gluconeogenesis. In addition, three new genes (two encoding putative enzymes and one hypothetical genes) whose depletion resulted in completely altered macrocolonies were also identified. The molecular mechanisms explaining the roles of these proteins in biofilm formation are currently under investigation.