6533b862fe1ef96bd12c6e37
RESEARCH PRODUCT
Comparison of CRISPR and marker based methods for the engineering of phage T7
Chrystala ConstantinidouMatthew TridgettAndrew D. MillardJohn N. DuncanAntonia P. SagonaAriadna Montero-blayPaul R. MacdonaldAlfonso JaramilloChristian HarrisonAurelija M. Grigonytesubject
0303 health sciences030306 microbiologyMutantComputational biologyBiologybiology.organism_classificationGenomeBacteriophage03 medical and health sciencesLytic cycleCRISPRHomologous recombinationGeneSelection (genetic algorithm)030304 developmental biologydescription
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 genescmkortrxhave 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.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2020-01-13 |