CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants
Year: 2025
Type: article
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-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 we 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.
Source: PLoS Pathogens
Authors: Celia Gil-Campillo, Johann Mignolet, Asier Domínguez-San Pedro, Beatriz Rapún-Araiz, Axel B. Janssen, Vincent de Bakker, Jan-Willem Veening, Junkal Garmendia
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,043
Funders: National Science Foundation, European Commission, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, Sociedad Española de Neumología y Cirugía Torácica, Gobierno de Navarra, Instituto de Salud Carlos III, Agencia Estatal de Investigación, HORIZON EUROPE Marie Sklodowska-Curie Actions
Awards: G1969249031, Bacteriocins and human commensal bacteria as a new strategy to inhibit the opportunistic human pathogen Streptococcus pneumoniae, G2364614043, G2374388372, G2608120745, Deciphering in vivo control of competence and transformation in Streptococcus pneumoniae, NCCR AntiResist: New approaches to combat antibiotic-resistant bacteria, G2730627974, G3332134968, G3748456914, G4338141805, Deciphering bacterial transformation: Control and bottlenecks in competence-dependent recombination, Construction of nanoscale devices using DNA self-assembly, scCRISPRi-seq and advanced microscopy to identify key virulence factors and heterogeneous host responses during Streptococcus pneumoniae infection, G6023213073, G7373938575, G7434936867, G8690818240
OpenAlex ID: W4415748418