Department of Medical Microbiology and Infection Control, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, Netherlands.
Department of Medical Microbiology and Infection Control, Amsterdam Institute of Infection & Immunity, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, Netherlands.
Tuberculosis (Edinb). 2020 Sep;124:101983. doi: 10.1016/j.tube.2020.101983. Epub 2020 Aug 12.
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.
过去几十年中，由于新型分子工具的出现，遗传工程致病性分枝杆菌的能力得到了显著提高。最近，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 干扰转化为分枝杆菌的高效基因组编辑工具。我们证明，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、精确的 DNA 断裂和插入缺失形成，而没有任何脱靶效应。此外，使用双 sgRNA，该系统可同时用于产生两个插入缺失或创建特定缺失。使用 CRISPR-Cas9 介导的基因编辑工具箱在结核分枝杆菌中进行单步操作的能力将加速对这种致命病原体的研究。
