A highly efficient identification of mutants generated by CRISPR/Cas9 using the non‑functional DsRed assisted selection in Aspergillus oryzae

World J Microbiol Biotechnol. 2021 Jul 9;37(8):132. doi: 10.1007/s11274-021-03100-8.


The CRISPR/Cas9 system has become a great tool for target gene knock-out in filamentous fungi. It is laborious and time-consuming that identification mutants from a large number of transformants through PCR or enzyme-cut method. Here, we first developed a CRISPR/Cas9 system in Aspergillus oryzae using AMA1-based autonomously replicating plasmid and Cas9 under the control of the Aspergillus nidulans gpdA promoter. By the genome editing technique, we successfully obtained mutations within each target gene in Aspergillus oryzae. Then, we put the protospacer sequence of a target gene and its protospacer adjacent motif (PAM) behind the start codon "ATG" of DsRed, yielding the non‑functional DsRed (nDsRed) reporter gene, and the nDsRed reporter gene could be rescued after successful targeted editing. Moreover, this method was also applied by targeting the kojic acid synthesis gene kojA, and the transformants with DsRed activity were found to harbor targeted mutations in kojA. These results suggest that the nDsRed can be used as a powerful tool to facilitate the identification of mutants generated by CRISPR/Cas9 in Aspergillus oryzae.

Keywords: Aspergillus oryzae; CRISPR/Cas9; DsRed; Identification mutant.

Publication types

  • Evaluation Study

MeSH terms

  • Aspergillus oryzae / enzymology
  • Aspergillus oryzae / genetics*
  • Aspergillus oryzae / metabolism
  • CRISPR-Cas Systems*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Genetic Techniques*
  • Luminescent Proteins / genetics*
  • Luminescent Proteins / metabolism
  • Mutation
  • Plasmids / genetics
  • Plasmids / metabolism
  • Pyrones / metabolism


  • Fungal Proteins
  • Luminescent Proteins
  • Pyrones
  • fluorescent protein 583
  • kojic acid