CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants

Garmendia, J.; Gil-Campillo, C.; Mignolet, J.; Dominguez-San Pedro, A.; Rapun-Araiz, B.; Janssen, A. B.; de Bakker, V.; Veening, J.-W.

doi:10.1101/2025.08.05.668841

bioRxiv · 10.1101/2025.08.05.668841

CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants

Garmendia, J.·Gil-Campillo, C.·Mignolet, J.·Dominguez-San Pedro, A.·Rapun-Araiz, B.·Janssen, A. B.·de Bakker, V.·Veening, J.-W.

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Abstract

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 hampered due to a limited molecular toolbox. To bridge this gap, we engineered a titratable anhydrotetracycline (aTc)-inducible CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) platform for efficient regulation of gene expression in H. influenzae. 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. We demonstrated that CRISPRi-programmed fitness defects can be rescuable, and refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, an extensive user-friendly online resource for visual inspection of H. influenzae genome annotations, including sgRNA spacers. The inducible 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. ImportanceCRISPRi-seq is a robust method to study bacterial gene fitness and essentiality via relative quantification and comparison of sgRNA abundance at a genome-wide scale. Here, we present a novel CRISPRi system for individual genes or pooled libraries knockdown in Haemophilus influenzae. A genome-wide CRISPRi library designed to cover 99.27% of all total genetic features in the genome of RdKW20 strain was constructed and screened in two laboratory growth media through CRISPRi-seq, uncovering growth medium-dependent fitness cost, further confirmed with individual knockdown/knockout mutants. We also introduce HaemoBrowse (https://HaemoBrowse.VeeningLab.com), through which genome annotations and sgRNA design on H. influenzae genomes can be readily inspected. This platform provides a valuable tool for gene function and essentiality analyses in a notorious human pathobiont.

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Garmendia, J., Gil-Campillo, C., Mignolet, J., Dominguez-San Pedro, A., Rapun-Araiz, B., Janssen, A. B., de Bakker, V., Veening, J.-W.. 2025-08-07. CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants. https://doi.org/10.1101/2025.08.05.668841

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