Development of an inducible CRISPR interference system for Haemophilus influenzae reveals genome-wide and medium growth determinants

Work in the human pathobiont Haemophilus influenzae has pioneered functional genomics in bacteria such as genome-wide transposon mutagenesis combined with deep sequencing. These approaches unveiled a large set of likely essential genes, but functional studies are lagging behind due to a limited molecular toolbox for studying essential genes. To bridge this gap, we engineered a titratable anhydrotetracycline (aTc)-inducible Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) platform for efficient regulation of gene expression in H. influenzae RdKw20. Genome-wide fitness analyses in two different in vitro culture media by CRISPRi-seq revealed growth medium-dependent fitness cost for a panel of H. influenzae genes, demonstrated that CRISPRi-programmed fitness defects can be rescueable, and refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, a comprehensive and user-friendly online resource for visually inspecting and exploring H. influenzae genome annotations and efficient sgRNA spacers. The CRISPRi platform described here represents a valuable tool enabling functional genomics and the study of essential genes, thereby contributing to the identification of therapeutic targets for developing drugs and vaccines against H. influenzae.