Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7
Aurelija M. Grigonyte,
Christian Harrison,
Paul R. MacDonald,
Ariadna Montero-Blay,
Matthew Tridgett,
John Duncan,
Antonia P. Sagona,
Chrystala Constantinidou,
Alfonso Jaramillo,
Andrew Millard
Affiliations
Aurelija M. Grigonyte
Warwick Integrative Synthetic Biology Centre (WISB) and School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
Christian Harrison
Department Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK
Paul R. MacDonald
MOAC DTC, Senate House, University of Warwick, Coventry CV4 7AL, UK
Ariadna Montero-Blay
EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr Aiguader 88, 08003 Barcelona, Spain
Matthew Tridgett
Warwick Integrative Synthetic Biology Centre (WISB) and School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
John Duncan
Warwick Integrative Synthetic Biology Centre (WISB) and School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
Antonia P. Sagona
Warwick Integrative Synthetic Biology Centre (WISB) and School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
Chrystala Constantinidou
Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
Alfonso Jaramillo
Warwick Integrative Synthetic Biology Centre (WISB) and School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
Andrew Millard
Department Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK
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.
