Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni.
CRISPRi
Campylobacter jejuni
gene regulation
Journal
BMC microbiology
ISSN: 1471-2180
Titre abrégé: BMC Microbiol
Pays: England
ID NLM: 100966981
Informations de publication
Date de publication:
05 10 2022
05 10 2022
Historique:
received:
28
04
2022
accepted:
15
09
2022
entrez:
5
10
2022
pubmed:
6
10
2022
medline:
12
10
2022
Statut:
epublish
Résumé
Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.
Sections du résumé
BACKGROUND
Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176.
RESULTS
The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM).
CONCLUSIONS
This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.
Identifiants
pubmed: 36199015
doi: 10.1186/s12866-022-02645-4
pii: 10.1186/s12866-022-02645-4
pmc: PMC9533551
doi:
Substances chimiques
Arylsulfatases
EC 3.1.6.1
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
238Subventions
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
Pays : United Kingdom
Informations de copyright
© 2022. The Author(s).
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