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Updated: Aug 15, 2025
Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
Published on: October 18, 2022
Protocol for gene characterization in Aspergillus niger using 5S rRNA-CRISPR-Cas9-mediated Tet-on inducible promoter
Xiaomei Zheng1, Timothy Cairns2, Ping Zheng1
1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; University of Chinese Academy of Sciences, Beijing 100049, China; National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China.
Abstract:
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.
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