5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger.,ACS Synthetic Biology

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5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-04-30 , DOI: 10.1021/acssynbio.7b00456
Xiaomei Zheng _{1,

2} , Ping Zheng _{1,

2} , Kun Zhang _{1,

2,

3} , Timothy C Cairns ₄ , Vera Meyer ₄ , Jibin Sun _{1,

2} , Yanhe Ma ₁

Affiliation

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 5S 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 B1. 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.

中文翻译：

用于指导 RNA 表达的 5S rRNA 启动子可在黑曲霉中实现高效 CRISPR/Cas9 基因组编辑。

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表达启动子是一项重大的技术挑战。在这里，我们使用工业上重要的真菌黑曲霉来证明5S rRNA基因在真核生物中高度保守且高效表达，可以用作指导RNA启动子。使用短（40-bp）同源供体DNA，在数十个转化体中建立了基因编辑系统，其精确基因修饰率达到100%。该系统也适用于设计染色体的生成，如霉菌毒素伏马菌素 B1 生物合成所需的 48 kb 基因簇的删除所证明的那样。此外，该系统还促进了黑曲霉中多个基因的同时诱变。我们预计，使用 5S rRNA 基因作为向导 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，这里报道的系统将能够在模型和工业上重要的真菌中开发设计师染色体。

更新日期：2018-04-24

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