Protocol for gene characterization in Aspergillus niger using 5S rRNA-CRISPR-Cas9-mediated Tet-on inducible promoter exchange

This protocol presents an efficient genetic strategy to investigate gene function in the fungus Aspergillus niger. We combined 5S rRNA-CRISPR-Cas9 technology with Tet-on gene switch to generate conditional-expression mutants via precisely replacing native promoter with inducible promoter. We describe the design and DNA preparation for sgRNAs and donor DNA. We then detail the steps for DNA co-transformation into A. niger protoplasts by PEG-mediated transformation, followed by homozygote isolation. Finally, we describe the genome verification and strain validation of the isolates. For complete details on the use and execution of this protocol, please refer to Zheng et al. (2019).1.