CRISPR/Cpf1-mediated mutagenesis and gene deletion in industrial filamentous fungi Aspergillus oryzae and Aspergillus sojae
CRISPR/Cpf1-mediated mutagenesis and gene deletion in industrial filamentous fungi Aspergillus oryzae and Aspergillus sojae
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DOI：
10.1016/j.jbiosc.2021.12.017
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发表时间：
2022-03-08
影响因子：
2.8
通讯作者：
Maruyama, Jun-ichi
中科院分区：
文献类型：
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作者：
Katayama, Takuya;Maruyama, Jun-ichi
在工业应用中，如发酵和异源蛋白生产，各种米曲霉和大豆曲霉菌株被使用。尽管针对这些丝状真菌已经开发了基因工程技术，但将这种经典技术应用于许多菌株是困难的。因此，需要建立适用于各种工业菌株的创新技术。我们之前开发了一种使用CRISPR/Cas9系统的基因组编辑技术，用于水稻芽孢杆菌的高效基因工程；然而，该系统受到其原间隔邻近基序序列的限制。在大豆中，没有开发出使用基因组编辑的基因工程。在本研究中，我们旨在开发一种利用Cpf1核酸酶进行水稻芽孢杆菌和大豆芽孢杆菌基因工程的基因组编辑技术。构建基于ama1的基因组编辑载体，携带密码子优化的cpf1表达盒，将引导RNA表达盒插入cpf1基因组编辑载体中。利用合成的质粒，我们对稻瘟病菌的AowA和sC基因以及大豆的AswA基因进行了诱变。我们通过共同引入Cpf1基因组编辑质粒和供体质粒来删除这些基因。本研究表明，CRISPR/Cpf1系统可作为一种有效的替代CRISPR/Cas9系统对稻瘟病菌进行基因工程改造，为稻瘟病菌的高效基因工程提供了一种新的途径。(c) 2022，日本生物技术学会。版权所有。
In industrial applications such as fermentation and heterologous protein production, various Aspergillus oryzae and A. sojae strains are used. Although genetic engineering techniques have been developed for these filamentous fungi, applying such classical techniques to many strains is difficult. Therefore, the establishment of innovative technologies applicable to various industrial strains is required. We previously developed a genome editing technology using the CRISPR/Cas9 system for the efficient genetic engineering of A. oryzae; however, this system is limited by its protospacer adjacent motif sequence. In A. sojae, no genetic engineering using genome editing has been developed. In this study, we aimed to develop a genome editing technology using the Cpf1 nuclease for the genetic engineering of A. oryzae and A. sojae. AMA1-based genome editing vectors bearing codon-optimized cpf1 expression cassettes were constructed, and guide RNA expression cassettes were inserted into the Cpf1 genome editing vectors. Using the resultant plasmids, we performed mutagenesis of the AowA and sC genes in A. oryzae and the AswA gene in A. sojae. We deleted these genes by co-introducing the Cpf1 genome editing plasmid and the donor plasmid. Our study demonstrates that the CRISPR/Cpf1 system can be used as an efficient alternative to the CRISPR/Cas9 system to genetically engineer A. oryzae and as a new approach for efficient genetic engineering of A. sojae. (c) 2022, The Society for Biotechnology, Japan. All rights reserved.