Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V.

Details

Serval ID
serval:BIB_76DFAD337B53
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V.
Journal
Applied and environmental microbiology
Author(s)
Synefiaridou D., Veening J.W.
ISSN
1098-5336 (Electronic)
ISSN-L
0099-2240
Publication state
Published
Issued date
26/02/2021
Peer-reviewed
Oui
Volume
87
Number
6
Pages
e02762-20
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: epublish
Abstract
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.
Keywords
CRISPR, Cas9, Streptococcus pneumoniae, genome editing, plasmids
Pubmed
Web of science
Open Access
Yes
Create date
11/01/2021 14:58
Last modification date
27/03/2021 7:32
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