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A CRISPR‐Cas9 tool to explore the genetics of Bacillus subtilis phages
Author(s) -
Otte K.,
Kühne N.M.,
Furrer A.D.,
Baena Lozada L.P.,
Lutz V.T.,
Schilling T.,
Hertel R.
Publication year - 2020
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.13349
Subject(s) - crispr , bacillus subtilis , cas9 , guide rna , biology , trans activating crrna , mutagenesis , crispr interference , computational biology , streptococcus pyogenes , dna , genetics , genome editing , oligonucleotide , gene , mutation , bacteria , staphylococcus aureus
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.