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A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages

  • Here we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built-up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.

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Author: Kolja Otte, Nora Kühne, Alexandra Furrer, Lina Lozada, Veronika Lutz, Tobias Schilling, Robert HertelORCiD
URL:https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/lam.13349
DOI:https://doi.org/10.1111/lam.13349
ISSN:1472-765X
Title of the source (English):Letters in Applied Microbiology
Document Type:Scientific journal article peer-reviewed
Language:English
Year of publication:2020
Volume/Year:71
Issue number:6
First Page:588
Last Page:595
Faculty/Chair:Fakultät 2 Umwelt und Naturwissenschaften / FG Synthetische Mikrobiologie
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