Abstract
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.
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Acknowledgements
We thank Dr. Van-Tuan Tran for kindly providing the plasmids pEX1 and pEX2B. This work was supported by Natural Science Foundation of China (32000049), Jiangxi Provincial Natural Science Foundation (20202BABL213042), Science and Technology Research Project of Jiangxi Provincial Department of Education (GJJ190611), and Doctoral Scientific Research Foundation of Jiangxi Science and Technology Normal University (2018BSQD030).
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Li, Y., Zhang, H., Fan, J. et al. A highly efficient identification of mutants generated by CRISPR/Cas9 using the non‑functional DsRed assisted selection in Aspergillus oryzae. World J Microbiol Biotechnol 37, 132 (2021). https://doi.org/10.1007/s11274-021-03100-8
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DOI: https://doi.org/10.1007/s11274-021-03100-8