Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes
Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes
复制标题
使用改进的可回收 CRISPR-HDR 工具箱对黑曲霉进行高效基因组编辑及其在引入异源基因多拷贝中的应用
DOI：
10.1016/j.mimet.2019.105655
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发表时间：
2019-08-01
影响因子：
2.2
通讯作者：
Pan, Li
中科院分区：
文献类型：
--
作者：
Dong, Hongzhi;Zheng, Junwei;Pan, Li
黑曲霉因其生产胞外分泌蛋白的高能力而成为重要的酶工业生产者。 CRISPR/Cas9 系统已被开发为黑曲霉的基因操作工具。然而，黑曲霉仅开发了 CRISPR/Cas9 系统的基本功能，例如 Cas9 核酸酶的密码子优化和向导 RNA (gRNA) 表达的启动子筛选。用于操作大基因组片段和多基因敲入的 CRISPR/Cas9 系统仍需建立。在这里，我们基于供体DNA（dDNA）和含有AMA1和pyrG标记的质粒改进了CRISPR/Cas9同源直接修复（CRISPR-HDR）工具箱，从而允许回收pyrG和Cas9组件。此外，我们使用 CRISPR-HDR 工具箱敲除 0 kb（仅原型间隔子）、2 kb、10 kb 甚至 50 kb 基因片段。该 CRISPR-HDR 工具箱还可用于同时敲入两种高表达的细胞外分泌蛋白——葡糖淀粉酶 A (glaA) 和 α-淀粉酶（amyA，两个拷贝）的基因座上的多个基因。在我们的研究中，两个或三个拷贝的葡萄糖氧化酶（goxC）被精确地敲入amyA和glaA的位点，导致酶活性增加4倍（869.86 U/mL）。这个CRISPR-HDR工具箱可以很容易地操作，并且基于AMA1的质粒可以在5-氟乳清酸和尿苷的选择性压力下轻松去除。
Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger. However, only the basic functions of the CRISPR/Cas9 system, such as codon optimization of Cas9 nucleases and promoter screening of guide RNA (gRNA) expression, have been developed in A. niger. The CRISPR/Cas9 system for manipulating large genomic fragments and multiple gene knock-ins still needs to be established. Here, we improved the CRISPR/Cas9 homologous direct repair (CRISPR-HDR) tool box based on donor DNAs (dDNAs) and plasmid harboring AMA1 and the pyrG marker, allowing recycling of pyrG and Cas9 components. Furthermore, we used the CRISPR-HDR tool box to knock out the 0 kb (protospacer only), 2 kb, 10 kb and even 50 kb gene fragments. This CRISPR-HDR tool box could also be used to simultaneously knock in multiple genes at the loci of two highly expressed extracellular secreted proteins, glucoamylase A (glaA) and alpha-amylase (amyA, two copies). In our study, two or three copies of glucose oxidase (goxC) were precisely knocked in at the loci of amyA and glaA, resulting in 4-fold increased enzyme activity (869.86 U/mL). This CRISPR-HDR tool box can be easily manipulated, and the AMA1-based plasmid can be easily removed under selective pressure of 5-fluoroorotic acid and uridine.