Novel CRISPR/Cas system for highly efficient genome editing in A. niger

The filamentous fungus Aspergillus niger (A. niger) is known for its high capacity for protein secretion. CRISPR/Cas-based systems are currently being used to generate high-yielding A. niger production strains. Given the difficult patent situation for the CRISPR nucleases known and used so far, limiting commercial use, we identified new Cas nucleases for genome editing based on metagenomic DNA analyses from environmental samples. The generated dataset allowed us to identify several novel nucleases, so-called G-dase M, with little or no homology to Cas proteins from public databases. To obtain a proprietary CRISPR tool for strain development of A. niger, a previously characterized G-dase M nuclease was established for genome editing.

We have successfully shown that the selected G-dase M nuclease can be programmed to efficiently and precisely introduce DNA double-strand breaks into the A. niger genome. An AMA1-based plasmid was adapted for optimized G-dase M expression and tRNA-gRNA transcription in A. niger. The functionality of the novel CRISPR/G-dase M single plasmid system was evaluated by clone reduction assays for different chromosomal genes in the NHEJ-deficient A. niger host strain. Furthermore, CRISPR/G-dase M activity induced HDR-mediated gene-specific integration of a reporter gene into various chromosomal targets with an efficiency of up to 99 %. In addition, we have developed a system to reduce the metabolic burden of plasmid-based overexpression of the nuclease by uncoupling the expression of G-dase M and tRNA-gRNA. For this purpose, the G‑dase M expression cassette was integrated into the genome of A. niger, while the regulatory elements for tRNA-gRNA transcription were provided on the AMA1-based plasmid. This strategy effectively reduced the metabolic burden caused by overexpression of the episomal expressed nuclease.

In summary, we have developed a highly efficient CRISPR system for genome editing in A. niger using the novel class 2 type V G-dase M nuclease. These results have shown the functionality of our proprietary CRISPR nuclease G-dase M in A. niger and will thus expand the molecular toolbox for genetic modification of filamentous fungi.