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

Zheng, Xiaomei; Cairns, Timothy; Zheng, Ping; Meyer, Vera; Sun, Jibin

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.
Published in: STAR Protocols, 10.1016/j.xpro.2022.101838, Elsevier