@misc{OtteKuehneFurreretal., author = {Otte, Kolja and K{\"u}hne, Nora and Furrer, Alexandra and Lozada, Lina and Lutz, Veronika and Schilling, Tobias and Hertel, Robert}, title = {A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages}, series = {Letters in Applied Microbiology}, volume = {71}, journal = {Letters in Applied Microbiology}, number = {6}, issn = {1472-765X}, doi = {10.1111/lam.13349}, pages = {588 -- 595}, abstract = {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.}, language = {en} }