Comparison of CRISPR and marker-based methods for the engineering of phage T7

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• dc.contributor.author Grigonyte, Aurelija M.
• dc.contributor.author Harrison, Christian
• dc.contributor.author MacDonald, Paul R.
• dc.contributor.author Montero-Blay, Ariadna, 1994-
• dc.contributor.author Tridgett, Matthew
• dc.contributor.author Duncan, John S.
• dc.contributor.author Sagona, Antonia P.
• dc.contributor.author Constantinidou, Chrystala
• dc.contributor.author Jaramillo, Alfonso
• dc.contributor.author Millard, Andrew
• dc.date.accessioned 2020-04-28T10:42:58Z
• dc.date.available 2020-04-28T10:42:58Z
• dc.date.issued 2020
• dc.description.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.
• dc.description.sponsorship A.M.G. is funded by a doctoral fellowship from the EPSRC & BBSRC Centre for Doctoral Training in Synthetic Biology (grant EP/L016494/1). P.R.M is funded by a doctoral fellowship from the EPSRC Doctoral Training Centre Molecular Organisation and Assembly in Cells (Grant No. EP/F500378/1). C.H. is funded by a PhD scholarship from the Dept Genetics and Genome Biology, University of Leicester. A.M.B, A.P.S. were funded by the European Commission grant (FP7-ICT-610730, EVOPROG) to A.J. J.D. was funded by the European Commission grant (FP7-KBBE-613745, PROMYS) to A.J. A.J. is funded by the European Commission grants (FP7-ICT-610730, EVOPROG; FP7-KBBE-613745, PROMYS; H2020-MSC-642738, MetaRNA), the BBSRC grant EVO-ENGINE BB/P020615/1, FP7-KBBE (no 613745, PROMYS), and EPSRC-BBSRC (BB/M017982/1, WISB center). A.M. is funded by MRC (MR/L015080/1) and NERC (NE/N019881/1)
• dc.format.mimetype application/pdf
• dc.identifier.citation Grigonyte AM, Harrison C, MacDonald PR, Montero-Blay A, Tridgett M, Duncan J et al. Comparison of CRISPR and marker-based methods for the engineering of phage T7. Viruses. 2020 Feb 10;12(2). pii: E193. DOI: 10.3390/v12020193
• dc.identifier.doi http://dx.doi.org/10.3390/v12020193
• dc.identifier.issn 1999-4915
• dc.identifier.uri http://hdl.handle.net/10230/44362
• dc.language.iso eng
• dc.publisher MDPI
• dc.relation.ispartof Viruses. 2020 Feb 10;12(2). pii: E193
• dc.relation.projectID info:eu-repo/grantAgreement/EC/FP7/610730
• dc.relation.projectID info:eu-repo/grantAgreement/EC/FP7/613745
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/642738
• dc.rights © 2020 Aurelija M. Grigonyte et al. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.other Bacteriòfags
• dc.subject.other Genomes
• dc.subject.other Genètica
• dc.title Comparison of CRISPR and marker-based methods for the engineering of phage T7
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion