A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages,Letters in Applied Microbiology

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A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages
Letters in Applied Microbiology ( IF 2.0 ) Pub Date : 2020-09-15 , DOI: 10.1111/lam.13349
K Otte ₁ , N M Kühne ₁ , A D Furrer ₁ , L P Baena Lozada ₁ , V T Lutz ₁ , T Schilling ₁ , R Hertel _{1,

2}

Affiliation

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.

中文翻译：

探索枯草芽孢杆菌噬菌体遗传学的 CRISPR-Cas9 工具

在此，我们推出 pRH030，这是一种用于芽孢杆菌噬菌体等基因工程的新型 CRISPR-Cas9 工具。它基于化脓性链球菌 cas9 及其天然组成型启动子、tracrRNA 和 gRNA 前体。 Cas9的组成型表达有利于病毒攻击者的灭活，并将噬菌体诱变效率提高至100%。 gRNA 前体可以构建为人工 CRISPR 阵列，该阵列具有由普通寡核苷酸组装而成的最多 5 个间隔区（靶序列），并直接克隆到 pRH030 中。所需的时间和资源与单个 gRNA 克隆相当。这些特性使 pRH030 成为一种有吸引力的芽孢杆菌噬菌体修饰新系统，并使其有资格用于基因构建以外的研究。

更新日期：2020-09-15

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