基于5S rRNA的黑曲霉CRISPR-Cas9高效多位点无痕基因组编辑系统研究

黑曲霉是有机酸、工业用酶与医药蛋白的重要生产菌株，但缺乏高效遗传操作工具导致其生产潜能的开发利用受到严重制约。CRISPR-Cas9系统作为新一代基因组编辑技术，为多种生物基因组定向改造与调控带来革命性突破。有报道尝试在黑曲霉中建立CRISPR-Cas9系统，但效率相对较低。其关键难题是真核生物中缺乏普适性的sgRNA高效表达方法。我们首次提出以5S rRNA基因为启动子起始sgRNA的表达，实验证实其引导Cas9对黑曲霉基因组定点切割效率可达100%。以此为基础，本项目将借助双向筛选标记的使用、供体DNA的无痕设计与NHEJ暂时性失活的结合，建立黑曲霉无痕基因组编辑技术；并进一步优化靶序列的选择、多基因sgRNA比例、供体DNA同源臂长度与用量等实现多基因多位点的快速编辑，总结形成黑曲霉基因组编辑的推荐方法，大幅降低黑曲霉基因编辑难度，为推动黑曲霉基础研究和工业菌种改造提供重要技术支撑。

Aspergillus niger is a very important industrial workhouse used for the production of organic acids, industrial enzymes and pharmaceutical proteins. Lack of efficient genetic manipulation tools dramatically restricts the full exploitation of its industrial potential. As a new generation of genome editing technology, CRISPR-Cas9 system brings a revolutionary breakthrough in targeted genome modification for many organisms. The adaption of CRISPR-Cas9 for A. niger was also tried but inefficient. The major technical obstacle is the lack of generally applicable sgRNA expression approach, and all the examples reported were tested to be less efficient in A. niger. In the previous study, we at the first time proposed to use well-recognized 5S rRNA for the sgRNA expression, in which the gene disruption frequency reached up to nearly 100%. Based on this invention, the project will further develop a rapid marker-free CRISPR-Cas9 system in combination with dual selection markers, marker-free donor DNA and NHEJ temporarily deficient chassis. Moreover, we will further optimize the targeting site, length of homology arm of the donor DNA and the ratio of sgRNA and donor DNA to establish a standardized protocol for highly efficient multiple gene targeting. The invention of this versatile CRISPR-Cas9 system for A. niger will greatly facilitate fundamental study and development of cell factory in A. niger.