5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger
5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger
复制标题
用于引导 RNA 表达的 5S rRNA 启动子在黑曲霉中实现高效 CRISPR/Cas9 基因组编辑
DOI：
10.1021/acssynbio.7b00456
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发表时间：
2019-07-01
影响因子：
4.7
通讯作者：
Ma, Yanhe
中科院分区：
文献类型：
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作者：
Zheng, Xiaomei;Zheng, Ping;Ma, Yanhe
CRISPR/Cas9系统是一种革命性的基因组编辑工具。然而，在真核生物中，寻找和优化合适的引导RNA表达启动子是一个重大的技术挑战。在这里，我们使用工业上重要的真菌黑曲霉，证明了SS rRNA基因在真核生物中既高度保守又高效表达，可以作为引导RNA启动子。利用短的（40-bp）同源供体DNA对数十个转化子进行了100%的精确基因修饰。该系统也适用于设计染色体的产生，如真菌毒素伏马菌素（fumonisin Bl）生物合成所需的48 kb基因簇的缺失所证明的那样。此外，该系统还促进了黑曲霉多个基因的同时诱变。我们预计，利用5S rRNA基因作为引导RNA启动子可以广泛应用于工程高效的真核CRISPR/Cas9工具包。此外，这里报道的系统将使设计染色体在模型和工业上重要的真菌的发展。
The CRISPR/Cas9 system is a revolutionary genome editing tool. However, in eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Here we used the industrially important fungus, Aspergillus niger, to demonstrate that the SS rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for generation of designer chromosomes, as evidenced by deletion of a 48 kb gene cluster required for biosynthesis of the mycotoxin fumonisin Bl. Moreover, this system also facilitated simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable development of designer chromosomes in model and industrially important fungi.