An efficient marker-free genome editing method for <i>Aspergillus niger</i>.

Yuyu SHEN; Zhongxiu CHEN; Jie CHEN; Baoding ZHAO; Jia LÜ; Ling GUI; Fuping LU; Ming LI

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An efficient marker-free genome editing method for Aspergillus niger.

Author: Yuyu SHEN ¹ ; Zhongxiu CHEN ¹ ; Jie CHEN ¹ ; Baoding ZHAO ¹ ; Jia LÜ ¹ ; Ling GUI ¹ ; Fuping LU ¹ ; Ming LI ¹
Author Information

1. Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science and Technology), Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
Publication Type:Journal Article
Keywords: Aspergillus niger; CRISPR/Cas9; genome editing; homologous recombination; marker-free; non-homologous end-joining (NHEJ)
MeSH: Gene Editing; Aspergillus niger/genetics*; CRISPR-Cas Systems/genetics*; Plasmids/genetics*
From: Chinese Journal of Biotechnology 2022;38(12):4744-4755
CountryChina
Language:Chinese
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.