Efficient gene deletion and replacement in Aspergillus niger by modified in vivo CRISPR/Cas9 systems

Aspergillus niger , as an important industrial strain, is widely used in the production of a variety of organic acids and industrial enzymes. To excavate the greater potential of A. niger as a cell factory, the development of highly efficient genome editing techniques is crucial. Here, we developed a modified CRISPR/Cas9 system for A. niger highlighted in two aspects: (1) construction of a single and easy-to-use CRISPR/Cas9 tool plasmid derived from pAN7-1 which is widely used in filamentous fungi; (2) redesign of the easy-to-switch “ribozyme–gRNA–ribozyme (RGR)” element in the tool plasmid. We examined the gene inactivation efficiency without repair fragment and the gene replacement efficiency with repair fragment utilizing the modified system, respectively, and both of them reach the efficiency as high as over 90%. Especially, the co-transformation of the tool plasmid and the specific repair fragment can easily realize one-step knock-out/knock-in of target genes, even with the length of homologous arms as only 100 bp. The establishment of this system will lay a solid foundation for the gene function research and rational design of cell factory in A. niger or broader filamentous fungi hosts.