Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae
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To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins.
To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations.
We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.
KeywordsAspergillus oryzae CRISPR/Cas9 system Filamentous fungi Genome editing
This work was supported by a Grant-in-Aid for Young Scientists from the Japan Society for the Promotion of Science. Funding for this study was also provided by Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries from National Agriculture and Food Research Organization, Japan.
Supplementary Methods—DNA manipulation, Media, and Preparation of protein extracts and western blot analysis.
Supplementary Material—Sequences of sgRNA, U6 promoter, U6 terminator, and codon-optimized cas9.
Supplementary Table 1—Primers used in this study.
Supplementary Fig. 1—Designation of protospacer sequences for the wA mutagenesis.
Supplementary Fig. 2—Sequencing analyses of the target sequence within the wA gene in two independent wA mutants.