CRISPR-Cas9 has proven as a very powerful gene editing tool for Actinomyces, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes Streptomyces coelicolor M145 and Streptomyces lividans TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in Actinomyces.

中文翻译：

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对链霉菌中有效CRISPR-Cas9介导的重组进行Cas9表达水平的调节。

CRISPR-Cas9已被证明是放线菌的非常强大的基因编辑工具，可在这些生物技术相关微生物的选定菌株中进行精确而精确的基因组编辑。然而，由于其在未经测试的菌株中应用该系统的无效性，其在放线菌中的一般应用受到限制。在这里，我们提供了Cas9水平如何对模型放线菌天蓝色链霉菌M145和淡紫色链霉菌TK24有毒的证据，它们显示出生长延迟或缺乏生长。我们通过降低Cas9水平克服了这种毒性，并产生了一组质粒，其中Cas9的表达受茶碱诱导型或组成型启动子的控制。我们使用甘油摄取操纵子和放线菌素生物合成基因簇验证了这些CRISPR-Cas9系统的靶向性。