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. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

We established an in vitro clustered regularly interspaced short palindromic repeats (CRISPR)‐associated RNA‐guided DNA endonucleases (Cas9) system to efficiently produce specific genome editing in Aspergillus niger, using a novel recyclable, bidirectional selection marker gene amdS without the need of prior production of an amdS mutant. The donor DNA plasmid consisted of amdS open reading frame, promoter, terminator, and directional repeats (DRs) flanking sequences. It was cotransformed with recombinant nuclease Cas9 and the sgRNA, which targets to the pigment gene olvA of A. niger strain CBS513.88. The positive olive transformants, other than the wild‐type strain, were able to grow on the media containing acetamide as the sole nitrogen source and cesium chloride. Furthermore, culturing the transformants on media with fluoroacetamide and urea allowed a loop‐out of the amdS expression cassette by recombining the flanking DRs. This study confirmed the facts that the endogenous amdS can be used as a dominant marker and that it can be removed by counter‐selection in gene editing of A. niger. The proposed in vitro CRISPR/Cas9 method offers a powerful tool for marker‐free genetic manipulation of filamentous fungi industrial‐specific strains.