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Keywords = vB_BsuP-Goe1

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1 pages, 1098 KiB  
Article
A CRISPR-Cas9-Based Toolkit for Fast and Precise In Vivo Genetic Engineering of Bacillus subtilis Phages
by Tobias Schilling, Sascha Dietrich, Michael Hoppert and Robert Hertel
Viruses 2018, 10(5), 241; https://doi.org/10.3390/v10050241 - 4 May 2018
Cited by 37 | Viewed by 8468
Abstract
Phages are currently under discussion as a solution for the antibiotic crisis, as they may cure diseases caused by multi-drug-resistant pathogens. However, knowledge of phage biology and genetics is limited, which impedes risk assessment of therapeutic applications. In order to enable advances in [...] Read more.
Phages are currently under discussion as a solution for the antibiotic crisis, as they may cure diseases caused by multi-drug-resistant pathogens. However, knowledge of phage biology and genetics is limited, which impedes risk assessment of therapeutic applications. In order to enable advances in phage genetic research, the aim of this work was to create a toolkit for simple and fast genetic engineering of phages recruiting Bacillus subtilis as host system. The model organism B. subtilis represents a non-pathogenic surrogate of its harmful relatives, such as Bacillus anthracis or Bacillus cereus. This toolkit comprises the application CutSPR, a bioinformatic tool for rapid primer design, and facilitates the cloning of specific CRISPR-Cas9-based mutagenesis plasmids. The employment of the prophage-free and super-competent B. subtilis TS01 strain enables an easy and fast introduction of specific constructs for in vivo phage mutagenesis. Clean gene deletions and a functional clean gene insertion into the genome of the model phage vB_BsuP-Goe1 served as proof of concept and demonstrate reliability and high efficiency. The here presented toolkit allows comprehensive investigation of the diverse phage genetic pool, a better understanding of phage biology, and safe phage applications. Full article
(This article belongs to the Section Bacterial Viruses)
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