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Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping
Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single exper...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131345/ https://www.ncbi.nlm.nih.gov/pubmed/27905467 http://dx.doi.org/10.1038/srep37938 |
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author | Müller, Vilhelm Rajer, Fredrika Frykholm, Karolin Nyberg, Lena K. Quaderi, Saair Fritzsche, Joachim Kristiansson, Erik Ambjörnsson, Tobias Sandegren, Linus Westerlund, Fredrik |
author_facet | Müller, Vilhelm Rajer, Fredrika Frykholm, Karolin Nyberg, Lena K. Quaderi, Saair Fritzsche, Joachim Kristiansson, Erik Ambjörnsson, Tobias Sandegren, Linus Westerlund, Fredrik |
author_sort | Müller, Vilhelm |
collection | PubMed |
description | Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples. |
format | Online Article Text |
id | pubmed-5131345 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-51313452016-12-15 Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping Müller, Vilhelm Rajer, Fredrika Frykholm, Karolin Nyberg, Lena K. Quaderi, Saair Fritzsche, Joachim Kristiansson, Erik Ambjörnsson, Tobias Sandegren, Linus Westerlund, Fredrik Sci Rep Article Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples. Nature Publishing Group 2016-12-01 /pmc/articles/PMC5131345/ /pubmed/27905467 http://dx.doi.org/10.1038/srep37938 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Müller, Vilhelm Rajer, Fredrika Frykholm, Karolin Nyberg, Lena K. Quaderi, Saair Fritzsche, Joachim Kristiansson, Erik Ambjörnsson, Tobias Sandegren, Linus Westerlund, Fredrik Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping |
title | Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping |
title_full | Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping |
title_fullStr | Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping |
title_full_unstemmed | Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping |
title_short | Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping |
title_sort | direct identification of antibiotic resistance genes on single plasmid molecules using crispr/cas9 in combination with optical dna mapping |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131345/ https://www.ncbi.nlm.nih.gov/pubmed/27905467 http://dx.doi.org/10.1038/srep37938 |
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