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

Lett Appl Microbiol. 2020 Dec;71(6):588-595. doi: 10.1111/lam.13349. Epub 2020 Sep 15.


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.

Keywords: Bacillus subtilis; CRISPR array; Cas9; cloning; engineering; isolation; phage; resistance.

MeSH terms

  • Bacillus Phages / genetics*
  • Bacillus Phages / physiology
  • Bacillus subtilis / virology*
  • CRISPR-Cas Systems*
  • Genetic Engineering
  • Mutagenesis
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism


  • RNA, Guide, CRISPR-Cas Systems