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Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V

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...

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Autores principales: Synefiaridou, Dimitra, Veening, Jan-Willem
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105017/
https://www.ncbi.nlm.nih.gov/pubmed/33397704
http://dx.doi.org/10.1128/AEM.02762-20
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author Synefiaridou, Dimitra
Veening, Jan-Willem
author_facet Synefiaridou, Dimitra
Veening, Jan-Willem
author_sort Synefiaridou, Dimitra
collection PubMed
description 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. IMPORTANCE Streptococcus 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.
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spelling pubmed-81050172021-05-10 Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V Synefiaridou, Dimitra Veening, Jan-Willem Appl Environ Microbiol Methods 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. IMPORTANCE Streptococcus 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. American Society for Microbiology 2021-02-26 /pmc/articles/PMC8105017/ /pubmed/33397704 http://dx.doi.org/10.1128/AEM.02762-20 Text en Copyright © 2021 Synefiaridou and Veening. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Methods
Synefiaridou, Dimitra
Veening, Jan-Willem
Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V
title Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V
title_full Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V
title_fullStr Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V
title_full_unstemmed Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V
title_short Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V
title_sort harnessing crispr-cas9 for genome editing in streptococcus pneumoniae d39v
topic Methods
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105017/
https://www.ncbi.nlm.nih.gov/pubmed/33397704
http://dx.doi.org/10.1128/AEM.02762-20
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