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Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System
Genome editing is an effective tool for the functional examination of bacterial genes and for live attenuated vaccine construction. Here, we report a method to edit the genomic DNA of Bacillus anthracis and Bacillus cereus using the clustered regularly interspaced short palindromic repeats (CRISPR)/...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736576/ https://www.ncbi.nlm.nih.gov/pubmed/31551942 http://dx.doi.org/10.3389/fmicb.2019.01932 |
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author | Wang, Yanchun Wang, Dongshu Wang, Xiaojing Tao, Haoxia Feng, Erling Zhu, Li Pan, Chao Wang, Bowen Liu, Chunjie Liu, Xiankai Wang, Hengliang |
author_facet | Wang, Yanchun Wang, Dongshu Wang, Xiaojing Tao, Haoxia Feng, Erling Zhu, Li Pan, Chao Wang, Bowen Liu, Chunjie Liu, Xiankai Wang, Hengliang |
author_sort | Wang, Yanchun |
collection | PubMed |
description | Genome editing is an effective tool for the functional examination of bacterial genes and for live attenuated vaccine construction. Here, we report a method to edit the genomic DNA of Bacillus anthracis and Bacillus cereus using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 system. Using two prophages in B. anthracis as targets, large-fragment deletion mutants were achieved with rates of 100 or 20%. In B. cereus, we successfully introduced precise point mutations into plcR, with phenotypic assays showing that the resulting mutants lost hemolytic and phospholipase enzyme activities similar to B. anthracis, which is a natural plcR mutant. Our study indicates that CRISPR/Cas9 is a powerful genetic tool for genome editing in the Bacillus cereus group, and can efficiently modify target genes without the need for residual foreign DNA such as antibiotic selection markers. This system could be developed for use in the generation of marker-free live anthrax vaccines or for safer construction of microbiological candidate-based recombinant B. cereus. |
format | Online Article Text |
id | pubmed-6736576 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-67365762019-09-24 Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System Wang, Yanchun Wang, Dongshu Wang, Xiaojing Tao, Haoxia Feng, Erling Zhu, Li Pan, Chao Wang, Bowen Liu, Chunjie Liu, Xiankai Wang, Hengliang Front Microbiol Microbiology Genome editing is an effective tool for the functional examination of bacterial genes and for live attenuated vaccine construction. Here, we report a method to edit the genomic DNA of Bacillus anthracis and Bacillus cereus using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 system. Using two prophages in B. anthracis as targets, large-fragment deletion mutants were achieved with rates of 100 or 20%. In B. cereus, we successfully introduced precise point mutations into plcR, with phenotypic assays showing that the resulting mutants lost hemolytic and phospholipase enzyme activities similar to B. anthracis, which is a natural plcR mutant. Our study indicates that CRISPR/Cas9 is a powerful genetic tool for genome editing in the Bacillus cereus group, and can efficiently modify target genes without the need for residual foreign DNA such as antibiotic selection markers. This system could be developed for use in the generation of marker-free live anthrax vaccines or for safer construction of microbiological candidate-based recombinant B. cereus. Frontiers Media S.A. 2019-08-27 /pmc/articles/PMC6736576/ /pubmed/31551942 http://dx.doi.org/10.3389/fmicb.2019.01932 Text en Copyright © 2019 Wang, Wang, Wang, Tao, Feng, Zhu, Pan, Wang, Liu, Liu and Wang. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Wang, Yanchun Wang, Dongshu Wang, Xiaojing Tao, Haoxia Feng, Erling Zhu, Li Pan, Chao Wang, Bowen Liu, Chunjie Liu, Xiankai Wang, Hengliang Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System |
title | Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System |
title_full | Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System |
title_fullStr | Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System |
title_full_unstemmed | Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System |
title_short | Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System |
title_sort | highly efficient genome engineering in bacillus anthracis and bacillus cereus using the crispr/cas9 system |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736576/ https://www.ncbi.nlm.nih.gov/pubmed/31551942 http://dx.doi.org/10.3389/fmicb.2019.01932 |
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