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

## TL;DR

The study introduces a CRISPRi system for Haemophilus influenzae to identify essential genes and their growth dependencies in different media.

## Contribution

A novel inducible CRISPRi platform and CRISPRi-seq method for functional genomics in Haemophilus influenzae is developed.

## Key findings

- CRISPRi-seq revealed medium-specific fitness costs for H. influenzae genes.
- The study refined previous essential gene studies using CRISPRi-programmed fitness defects.
- HaemoBrowse was introduced as a resource for genome annotations and sgRNA design.

## 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.

CRISPRi-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.