Titre
Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V.
Type
article
Institution
UNIL/CHUV/Unisanté + institutions partenaires
Périodique
Auteur(s)
Synefiaridou, D.
Auteure/Auteur
Veening, J.W.
Auteure/Auteur
Liens vers les personnes
Liens vers les unités
ISSN
1098-5336
Statut éditorial
Publié
Date de publication
2021-02-26
Volume
87
Numéro
6
Première page
e02762
Dernière page/numéro d’article
20
Peer-reviewed
Oui
Langue
anglais
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: epublish
Publication Status: epublish
Résumé
CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by the detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae Specifically, we demonstrate an approach for making targeted markerless gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) is exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid, which is temperature sensitive for replication, by growing it at the nonpermissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The approaches developed here could potentially be adapted for use with other Gram-positive bacteria.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing more than 1 million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts to identify new vaccine candidates. Here, we introduced an easy-to-use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The approaches used here will advance genome editing projects in this important human pathogen.
PID Serval
serval:BIB_76DFAD337B53
PMID
Open Access
Oui
Date de création
2021-01-11T12:58:09.494Z
Date de création dans IRIS
2025-05-21T01:00:01Z
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Nom
33397704_BIB_76DFAD337B53.pdf
Version du manuscrit
published
Licence
https://creativecommons.org/licenses/by/4.0
Taille
1.8 MB
Format
Adobe PDF
PID Serval
serval:BIB_76DFAD337B53.P001
URN
urn:nbn:ch:serval-BIB_76DFAD337B532
Somme de contrôle
(MD5):ecd7ab575353c99bc75bcb66fa965748