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Lett Appl Microbiol ; 71(6): 588-595, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32615024

RESUMO

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.


Assuntos
Fagos Bacilares/genética , Bacillus subtilis/virologia , Sistemas CRISPR-Cas , Fagos Bacilares/fisiologia , Engenharia Genética , Mutagênese , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
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