Development of a CRISPR/Cas9D10A Nickase (nCas9)-Mediated Genome Editing Tool in Streptomyces.
Development of a CRISPR/Cas9D10A Nickase (nCas9)-Mediated Genome Editing Tool in Streptomyces.
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DOI：
10.1021/acssynbio.3c00466
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发表时间：
2023-09
影响因子：
4.7
通讯作者：
Jia-Xiang Ma;Wen-Yan He;Hui-Min Hua;Qian Zhu;Guo-Song Zheng;Andrei A Zimin;Wen-Fang Wang;Yin-Hua Lu
中科院分区：
文献类型：
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作者：
Jia-Xiang Ma;Wen-Yan He;Hui-Min Hua;Qian Zhu;Guo-Song Zheng;Andrei A Zimin;Wen-Fang Wang;Yin-Hua Lu
链霉菌具有产生广泛用于农业和兽医/人类医学的大量生物活性天然产物（NP）的强大能力。最近开发的基于CRISPR/Cas9的基因组编辑工具极大地促进了链霉菌中目标NP过量生产的菌株改良以及新NP的发现。然而，CRISPR/Cas9对宿主表现出高毒性，限制了其在许多DNA转化效率低的链霉菌菌株中的应用。在这项研究中，我们在模式菌株天蓝色链霉菌M145中开发了一种低毒性的基于CRISPR/Cas 9D 10A切口酶（nCas 9）的基因组编辑工具。我们表明，在靶向sgRNA和Cas蛋白两者存在的情况下，利用nCas 9而不是Cas9显著降低了对宿主的毒性并极大地增强了细胞存活。使用该工具，我们实现了单个基因和基因簇的删除，效率分别为87-100和63- 87%，以及两个基因或基因簇的同时删除，效率分别为47和43%。nCas 9的编辑效率与Cas9介导的编辑工具相当。最后，基于nCas 9的编辑工具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/nCas 9的编辑工具为链霉菌的工程化提供了方便有效的遗传修饰系统，特别是那些DNA转化效率低的链霉菌。
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.