PT  - JOURNAL ARTICLE
AU  - Aurelija M. Grigonyte
AU  - Christian Harrison
AU  - Paul R. MacDonald
AU  - Ariadna Montero-Blay
AU  - Matthew Tridgett
AU  - John Duncan
AU  - Antonia P. Sagona
AU  - Chrystala Constantinidou
AU  - Alfonso Jaramillo
AU  - Andrew Millard
TI  - Comparison of CRISPR and marker based methods for the engineering of phage T7
AID  - 10.1101/2020.01.12.903492
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.01.12.903492
4099  - http://biorxiv.org/content/early/2020/01/13/2020.01.12.903492.short
4100  - http://biorxiv.org/content/early/2020/01/13/2020.01.12.903492.full
AB  - 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 trx 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 fiber 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.