一种高效无选择标记的黑曲霉基因组编辑方法

An efficient marker-free genome editing method for Aspergillus niger

摘要:

黑曲霉(Aspergillus niger)是一种重要的工业生产菌株，被广泛地应用于生产酶制剂和有机酸，但仍需要进行基因组改造提高它的应用潜力。CRISPR/Cas9技术是一种被广泛采用的黑曲霉基因组编辑技术，但由于需要在基因组中整合选择标记或基因编辑效率还有待提高，影响了其在工业菌株改造中的应用。本研究建立了一种基于CRISPR/Cas9技术的高效无选择标记的基因编辑方法。首先，利用5S rRNA启动子启动sgRNA的表达，构建了一个含有AMA1(autonomously maintained in Aspergillus)复制起始片段的sgRNA和Cas9共表达质粒；同时通过敲除kusA基因构建非同源末端连接(non-homologous end joining pathway,NHEJ)修复缺陷的高效同源重组菌株；最后利用含有AMA1片段质粒的不稳定性，通过无抗平板传代丢失含有sgRNA和Cas9共表达质粒。利用该方法，在采用同源臂长度仅为20bp的无选择标记供体DNA进行基因编辑时，基因编辑效率可达到100%。该方法为黑曲霉基因功能的研究和细胞工厂的构建奠定了基础。

关键词:黑曲霉;CRISPR/Cas9;无标记;基因组编辑;非同源末端连接;同源重组

Abstract:

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A.niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A.niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A.niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A.niger.

Key words:Aspergillus niger;CRISPR/Cas9;marker-free;genome editing;non-homologous end-joining (NHEJ);homologous recombination