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Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology

[Image: see text] Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the p...

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Autores principales: Gussak, Alex, Ferrando, Maria Laura, Schrama, Mels, van Baarlen, Peter, Wells, Jerry Mark
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10510748/
https://www.ncbi.nlm.nih.gov/pubmed/37602730
http://dx.doi.org/10.1021/acssynbio.3c00110
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author Gussak, Alex
Ferrando, Maria Laura
Schrama, Mels
van Baarlen, Peter
Wells, Jerry Mark
author_facet Gussak, Alex
Ferrando, Maria Laura
Schrama, Mels
van Baarlen, Peter
Wells, Jerry Mark
author_sort Gussak, Alex
collection PubMed
description [Image: see text] Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing.
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spelling pubmed-105107482023-09-21 Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology Gussak, Alex Ferrando, Maria Laura Schrama, Mels van Baarlen, Peter Wells, Jerry Mark ACS Synth Biol [Image: see text] Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing. American Chemical Society 2023-08-21 /pmc/articles/PMC10510748/ /pubmed/37602730 http://dx.doi.org/10.1021/acssynbio.3c00110 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Gussak, Alex
Ferrando, Maria Laura
Schrama, Mels
van Baarlen, Peter
Wells, Jerry Mark
Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology
title Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology
title_full Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology
title_fullStr Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology
title_full_unstemmed Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology
title_short Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology
title_sort precision genome engineering in streptococcus suis based on a broad-host-range vector and crispr-cas9 technology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10510748/
https://www.ncbi.nlm.nih.gov/pubmed/37602730
http://dx.doi.org/10.1021/acssynbio.3c00110
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