利用内源性I型和III型CRISPR-Cas系统对嗜热放线菌进行高效基因组编辑。

High-efficiency genome editing of an extreme thermophile using endogenous type I and type III CRISPR-Cas systems.

作者信息

Wang Jinting, Wei Junwei, Li Haijuan, Li Yingjun

机构信息

State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology Huazhong Agricultural University Wuhan China.

Shenzhen Institute of Nutrition and Health Huazhong Agricultural University Shenzhen China.

出版信息

mLife. 2022 Dec 7;1(4):412-427. doi: 10.1002/mlf2.12045. eCollection 2022 Dec.

Abstract

is an attractive species in the bioindustry due to its valuable natural products, abundant thermophilic enzymes, and promising fermentation capacities. However, efficient and versatile genome editing tools are not available for this species. In this study, we developed an efficient genome editing tool for HB27 based on its endogenous type I-B, I-C, and III-A/B CRISPR-Cas systems. First, we systematically characterized the DNA interference capabilities of the different types of the native CRISPR-Cas systems in HB27. We found that genomic manipulations such as gene deletion, mutation, and in situ tagging could be easily implemented by a series of genome-editing plasmids carrying an artificial self-targeting mini-CRISPR and a donor DNA responsible for the recombinant recovery. We also compared the genome editing efficiency of different CRISPR-Cas systems and the editing plasmids with donor DNAs of different lengths. Additionally, we developed a reporter gene system for based on a heat-stable β-galactosidase gene , and constructed an engineered strain with a high production capacity of superoxide dismutases by genome modification.

摘要

由于其有价值的天然产物、丰富的嗜热酶和有前景的发酵能力,它是生物产业中一个有吸引力的物种。然而,该物种没有高效且通用的基因组编辑工具。在本研究中,我们基于其内源I-B型、I-C型和III-A/B型CRISPR-Cas系统,为HB27开发了一种高效的基因组编辑工具。首先,我们系统地表征了HB27中不同类型天然CRISPR-Cas系统的DNA干扰能力。我们发现,诸如基因缺失、突变和原位标记等基因组操作可以通过一系列携带人工自我靶向微型CRISPR和负责重组恢复的供体DNA的基因组编辑质粒轻松实现。我们还比较了不同CRISPR-Cas系统以及带有不同长度供体DNA的编辑质粒的基因组编辑效率。此外,我们基于热稳定的β-半乳糖苷酶基因lacS开发了一个用于HB27的报告基因系统,并通过基因组修饰构建了一个具有高超氧化物歧化酶生产能力的工程菌株。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/abdc/10989782/373bb3df8a17/MLF2-1-412-g006.jpg

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