Ma Jia-Xiang, He Wen-Yan, Hua Hui-Min, Zhu Qian, Zheng Guo-Song, Zimin Andrei A, Wang Wen-Fang, Lu Yin-Hua
College of Life Sciences, Shanghai Normal University, Shanghai 200234, China.
G.K. Scriabin Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino 142290, Russia.
ACS Synth Biol. 2023 Oct 20;12(10):3114-3123. doi: 10.1021/acssynbio.3c00466. Epub 2023 Sep 18.
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 . However, CRISPR/Cas9 shows high toxicity to the host, limiting its application in many strains with a low DNA transformation efficiency. In this study, we developed a low-toxicity CRISPR/Cas9 nickase (nCas9)-based genome editing tool in the model strain 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 , 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.
链霉菌具有强大的能力，能够产生大量广泛应用于农业以及兽医学/人类医学的生物活性天然产物（NPs）。最近开发的基于CRISPR/Cas9的基因组编辑工具极大地促进了目标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转化效率低的菌株中的应用。在本研究中，我们在模式菌株M145中开发了一种低毒性的基于CRISPR/Cas9切口酶（nCas9）的基因组编辑工具。我们表明，在存在靶向sgRNA和Cas蛋白的情况下，使用nCas9而非Cas9可显著降低对宿主的毒性并大大提高细胞存活率。使用该工具，我们实现了单个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生产雷帕霉素的菌株的基因组编辑，在该菌株中CRISPR/Cas9无法很好地发挥作用。我们实现了三个测试基因的缺失，效率为27.2 - 30%。总体而言，基于CRISPR/nCas9的编辑工具为链霉菌的工程改造提供了一种方便且高效的遗传修饰系统，特别是对于那些DNA转化效率低的链霉菌。
