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Comparison of CRISPR and marker-based methods for the engineering of Phage T7
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Grigonyte, Aurelija, Harrison, Christian, Macdonald, Paul R., Montero-Blay, Ariadna, Tridgett, Matthew, Duncan, John, Sagona, Antonia P., Constantinidou, Chrystala, Jaramillo, Alfonso and Millard, Andrew D. (2020) Comparison of CRISPR and marker-based methods for the engineering of Phage T7. Viruses, 12 (2). e193. doi:10.3390/v12020193 ISSN 1999-4915.
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Official URL: https://doi.org/10.3390/v12020193
Abstract
With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.
Item Type: | Journal Article | ||||||||||||||||||||||||||||||||||||
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Subjects: | Q Science > QH Natural history Q Science > QR Microbiology |
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Chemistry Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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SWORD Depositor: | Library Publications Router | ||||||||||||||||||||||||||||||||||||
Library of Congress Subject Headings (LCSH): | Bacteriophages, CRISPR (Genetics) | ||||||||||||||||||||||||||||||||||||
Journal or Publication Title: | Viruses | ||||||||||||||||||||||||||||||||||||
Publisher: | MDPI | ||||||||||||||||||||||||||||||||||||
ISSN: | 1999-4915 | ||||||||||||||||||||||||||||||||||||
Official Date: | 10 February 2020 | ||||||||||||||||||||||||||||||||||||
Dates: |
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Volume: | 12 | ||||||||||||||||||||||||||||||||||||
Number: | 2 | ||||||||||||||||||||||||||||||||||||
Article Number: | e193 | ||||||||||||||||||||||||||||||||||||
DOI: | 10.3390/v12020193 | ||||||||||||||||||||||||||||||||||||
Status: | Peer Reviewed | ||||||||||||||||||||||||||||||||||||
Publication Status: | Published | ||||||||||||||||||||||||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | ||||||||||||||||||||||||||||||||||||
Date of first compliant deposit: | 13 February 2020 | ||||||||||||||||||||||||||||||||||||
Date of first compliant Open Access: | 26 February 2020 | ||||||||||||||||||||||||||||||||||||
RIOXX Funder/Project Grant: |
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