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A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection

The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA c...

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Autores principales: Wongpayak, Panjaporn, Meesungnoen, Orapan, Saejang, Somchai, Subsoontorn, Pakpoom
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428258/
https://www.ncbi.nlm.nih.gov/pubmed/34567840
http://dx.doi.org/10.7717/peerj.11996
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author Wongpayak, Panjaporn
Meesungnoen, Orapan
Saejang, Somchai
Subsoontorn, Pakpoom
author_facet Wongpayak, Panjaporn
Meesungnoen, Orapan
Saejang, Somchai
Subsoontorn, Pakpoom
author_sort Wongpayak, Panjaporn
collection PubMed
description The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.
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spelling pubmed-84282582021-09-24 A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection Wongpayak, Panjaporn Meesungnoen, Orapan Saejang, Somchai Subsoontorn, Pakpoom PeerJ Bioengineering The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering. PeerJ Inc. 2021-09-06 /pmc/articles/PMC8428258/ /pubmed/34567840 http://dx.doi.org/10.7717/peerj.11996 Text en © 2021 Wongpayak et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Bioengineering
Wongpayak, Panjaporn
Meesungnoen, Orapan
Saejang, Somchai
Subsoontorn, Pakpoom
A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection
title A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection
title_full A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection
title_fullStr A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection
title_full_unstemmed A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection
title_short A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection
title_sort highly effective and self-transmissible crispr antimicrobial for elimination of target plasmids without antibiotic selection
topic Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428258/
https://www.ncbi.nlm.nih.gov/pubmed/34567840
http://dx.doi.org/10.7717/peerj.11996
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