University of Ljubljana Biotechnical Faculty, Chair of Biotechnology, Microbiology and Food Safety, Jamnikarjeva ulica 101, Ljubljana, Ljubljana, SI 1000, Slovenia.
University of Ljubljana Biotechnical Faculty, Chair of Biotechnology, Microbiology and Food Safety, Jamnikarjeva ulica 101, Ljubljana, Ljubljana, SI 1000, Slovenia.
J Microbiol Methods. 2022 Sep;200:106545. doi: 10.1016/j.mimet.2022.106545. Epub 2022 Aug 1.
CRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to "escaper colonies" carrying unedited genotype. Our approach allows a simple detection of "escaper" phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.
CRISPR-Cas9 技术已成为链霉菌菌株遗传工程的一种有前途的工具。然而，在实际应用中，在开发稳健的编辑程序时，仍有许多技术障碍需要克服。在这里，我们开发了 CRISPR-Cas 工具包的扩展，这是一种简单可靠的 cas9 监测工具，其 cas9 核酸酶与β葡糖苷酸酶（gusA）可视报告基因进行转录融合。Cas9-SD-GusA 工具可在引入携带 CRISPR-Cas9 机制的质粒后，原位鉴定表达 Cas9 核酸酶的细胞。值得注意的是，当 Cas9-SD-GusA 系统在最佳条件下应用时，显示 GusA 活性的 100%的菌落携带目标基因型。相比之下，在不显示 GusA 活性的菌落中，cas9 序列显示出主要的重组事件，导致携带未经编辑基因型的“逃逸菌落”。我们的方法可以简单地检测到“逃逸”表型，并作为一种有效的 CRISPR-Cas9 优化工具。
