链霉菌中 CRISPR/Cas9D10A 切口酶 (nCas9) 介导的基因组编辑工具的开发,ACS Synthetic Biology

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链霉菌中 CRISPR/Cas9D10A 切口酶 (nCas9) 介导的基因组编辑工具的开发
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2023-09-18 , DOI: 10.1021/acssynbio.3c00466
Jia-Xiang Ma ₁ , Wen-Yan He ₁ , Hui-Min Hua ₁ , Qian Zhu ₁ , Guo-Song Zheng ₁ , Andrei A Zimin ₂ , Wen-Fang Wang ₁ , Yin-Hua Lu _{1,

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Affiliation

链霉菌具有很强的生产大量生物活性天然产物（NP）的能力，这些天然产物广泛用于农业和兽医/人类医学。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，我们在模型菌株天蓝色链霉菌M145 中开发了一种基于低毒性 CRISPR/Cas9 ^D10A切口酶 (nCas9) 的基因组编辑工具。我们发现，在同时存在靶向 sgRNA 和 Cas 蛋白的情况下，使用 nCas9 代替 Cas9 显着降低了对宿主的毒性，并大大提高了细胞存活率。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%，同时删除两个基因或基因簇的效率分别为 47% 和 43%。nCas9的编辑效率与Cas9介导的编辑工具相当。最后，基于nCas9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（Streptomyces rapamycinicus）的基因组编辑，在该菌株中CRISPR/Cas9不能很好地发挥作用。我们以 27.2-30% 的效率实现了三个测试基因的删除。总的来说，基于 CRISPR/nCas9 的编辑工具为链霉菌工程，特别是那些 DNA 转化效率较低的链霉菌工程提供了一种方便、高效的遗传修饰系统。

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Development of a CRISPR/Cas9D10A Nickase (nCas9)-Mediated Genome Editing Tool in Streptomyces

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/Cas9^D10A 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.

更新日期：2023-09-18

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