Viruses, Vol. 12, Pages 193: Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7

Viruses, Vol. 12, Pages 193: Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7
Viruses doi: 10.3390/v12020193
Authors:
Grigonyte
Harrison
MacDonald
Montero-Blay
Tridgett
Duncan
Sagona
Constantinidou
Jaramillo
Millard

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.

Source: Viruses - February 9, 2020 Category: Virology Authors: Grigonyte Harrison MacDonald Montero-Blay Tridgett Duncan Sagona Constantinidou Jaramillo Millard Tags: Article Source Type: research