Abstract：
Streptomycetes have a strong ability to produce a vast array of bioactive natural products (NPs) widely used in agriculture and veterinary/human medicine. The recently developed CRISPR/Cas9-based genome editing tools have greatly facilitated strain improvement for target NP overproduction as well as novel NP discovery in Streptomyces. However, CRISPR/Cas9 shows high toxicity to the host, limiting its application in many Streptomyces strains with a low DNA transformation efficiency. In this study, we developed a low-toxicity CRISPR/Cas9D10A nickase (nCas9)-based genome editing tool in the model strain Streptomyces coelicolor M145. We showed that in the presence of both targeting sgRNA and Cas proteins, utilization of nCas9 instead of Cas9 significantly reduced the toxicity to the host and greatly enhanced cell survival. Using this tool, we achieved deletion of single genes and gene clusters with efficiencies of 87-100 and 63-87%, and simultaneous deletion of two genes or gene clusters with efficiencies of 47 and 43%, respectively. The editing efficiency of nCas9 is comparable to that of the Cas9-mediated editing tool. Finally, the nCas9-based editing tool was successfully applied for genome editing in the industrial rapamycin-producing strain Streptomyces rapamycinicus, in which CRISPR/Cas9 cannot work well. We achieved the deletion of three tested genes with an efficiency of 27.2-30%. Collectively, the CRISPR/nCas9-based editing tool offers a convenient and efficient genetic modification system for the engineering of streptomycetes, particularly those with low DNA transformation efficiency.
摘要：
链霉菌具有很强的产生广泛用于农业和兽医/人类医学的生物活性天然产物(NPs)的能力。最近开发的基于CRISPR/Cas9的基因组编辑工具极大地促进了靶NP过量生产的菌株改进以及链霉菌中的新NP发现。然而,CRISPR/Cas9对宿主有很高的毒性,限制了其在许多链霉菌菌株中的应用，DNA转化效率较低。在这项研究中,我们开发了一种低毒性CRISPR/Cas9D10A切口酶(nCas9)为基础的基因组编辑工具,用于模型菌株天色链霉菌M145.我们表明，在存在靶向sgRNA和Cas蛋白的情况下，利用nCas9而不是Cas9显着降低了对宿主的毒性，并大大提高了细胞的存活率。使用这个工具，我们实现了单基因和基因簇的缺失，效率分别为87-100和63-87%，同时缺失两个基因或基因簇，效率分别为47％和43％，分别。nCas9的编辑效率与Cas9介导的编辑工具的编辑效率相当。最后,基于nCas9的编辑工具已成功应用于工业雷帕霉素生产菌株雷帕霉素链霉菌的基因组编辑，其中CRISPR/Cas9不能很好地工作。我们实现了三个测试基因的缺失，效率为27.2-30％。总的来说，基于CRISPR/nCas9的编辑工具为链霉菌的工程提供了方便高效的遗传修饰系统，特别是那些具有低DNA转化效率。
