The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR-Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference-mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen.

中文翻译：

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使用嗜热链球菌 CRISPR1-Cas9 对致病性分枝杆菌进行有效的基因组编辑

由于新分子工具的产生，过去几十年来基因工程致病性分枝杆菌的能力显着提高。最近，化脓性链球菌和嗜热链球菌 CRISPR-Cas9 系统在分枝杆菌中的应用使基因编辑和高效的 CRISPR 干扰介导的转录调控成为可能。在这里，我们将 CRISPR 干扰转化为一种有效的分枝杆菌基因组编辑工具。我们证明了嗜热链球菌 CRISPR1-Cas9 (Sth1Cas9) 在海分枝杆菌和结核分枝杆菌中具有功能，能够实现高效和精确的 DNA 断裂和插入缺失，而没有任何脱靶效应。此外，使用双 sgRNA，该系统可用于同时生成两个插入缺失或创建特定缺失。