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Viruses. 2018 May 4;10(5). pii: E241. doi: 10.3390/v10050241.

A CRISPR-Cas9-Based Toolkit for Fast and Precise In Vivo Genetic Engineering of Bacillus subtilis Phages.

Author information

1
Department of Genomic and Applied Microbiology & Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August-University Göttingen, 37077 Göttingen, Germany. tschill2@gwdg.de.
2
Department of Genomic and Applied Microbiology & Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August-University Göttingen, 37077 Göttingen, Germany. sdietri@gwdg.de.
3
Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University Göttingen, 37077 Göttingen, Germany. mhopper@gwdg.de.
4
Department of Genomic and Applied Microbiology & Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August-University Göttingen, 37077 Göttingen, Germany. rhertel@gwdg.de.

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 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.

KEYWORDS:

Bacillus subtilis TS01; CRISPR-Cas9; CutSPR; artificial; bacteriophage; phage; vB_BsuP-Goe1; virus

PMID:
29734705
PMCID:
PMC5977234
DOI:
10.3390/v10050241
[Indexed for MEDLINE]
Free PMC Article

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