RT Journal Article
SR Electronic
T1 Comparison of CRISPR and marker based methods for the engineering of phage T7
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.01.12.903492
DO 10.1101/2020.01.12.903492
A1 Grigonyte, Aurelija M.
A1 Harrison, Christian
A1 MacDonald, Paul R.
A1 Montero-Blay, Ariadna
A1 Tridgett, Matthew
A1 Duncan, John
A1 Sagona, Antonia P.
A1 Constantinidou, Chrystala
A1 Jaramillo, Alfonso
A1 Millard, Andrew
YR 2020
UL http://biorxiv.org/content/early/2020/01/13/2020.01.12.903492.abstract
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.