Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology
Abstract Filamentous fungi represent an invaluable source of pharmaceutically active compounds. The development of versatile methods to genetically manipulate filamentous fungi is of great value for improving the low yields of bioactive metabolites and expanding chemical diversity. The CRISPR-Cas9-b...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2017-08-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-10052-3 |
| _version_ | 1818428291111976960 |
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| author | Yan-Mei Zheng Fu-Long Lin Hao Gao Gen Zou Jiang-Wei Zhang Gao-Qian Wang Guo-Dong Chen Zhi-Hua Zhou Xin-Sheng Yao Dan Hu |
| author_facet | Yan-Mei Zheng Fu-Long Lin Hao Gao Gen Zou Jiang-Wei Zhang Gao-Qian Wang Guo-Dong Chen Zhi-Hua Zhou Xin-Sheng Yao Dan Hu |
| author_sort | Yan-Mei Zheng |
| collection | DOAJ |
| description | Abstract Filamentous fungi represent an invaluable source of pharmaceutically active compounds. The development of versatile methods to genetically manipulate filamentous fungi is of great value for improving the low yields of bioactive metabolites and expanding chemical diversity. The CRISPR-Cas9-based system has become a common platform for genome editing in a variety of organisms. However, recent application of this technology in filamentous fungi is limited to model strains, a versatile method for efficient gene disruption in different fungi is lacking. Here, we investigated the utility of the CRISPR-Cas9 system in a less-studied fungus Nodulisporium sp. (No. 65-12-7-1), and we have developed an efficient CRISPR-Cas9-based gene disruption strategy by simultaneous transformation of in vitro transcriptional gRNA and the linear maker gene cassette into the Cas9-expressing fungi. We found that the linear marker gene cassette could not only allow for selection of transformants, but also significantly enhance the gene disruption efficiency by inserting itself into the Cas9 cut site. Moreover, the above approach also demonstrated its efficiency in two other phylogenetically distinct strains Aspergillus oryzae NSAR1 and Sporormiella minima (No. 40-1-4-1) from two different classes of Ascomycota. These results suggested that a versatile CRISPR-Cas9-based gene disruption method in filamentous fungi was established. |
| first_indexed | 2024-12-14T14:59:17Z |
| format | Article |
| id | doaj.art-435d45484750440791e74b5b38caee94 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T14:59:17Z |
| publishDate | 2017-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-435d45484750440791e74b5b38caee942022-12-21T22:56:54ZengNature PortfolioScientific Reports2045-23222017-08-017111010.1038/s41598-017-10052-3Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technologyYan-Mei Zheng0Fu-Long Lin1Hao Gao2Gen Zou3Jiang-Wei Zhang4Gao-Qian Wang5Guo-Dong Chen6Zhi-Hua Zhou7Xin-Sheng Yao8Dan Hu9Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan UniversityInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan UniversityInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan UniversityCAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of SciencesInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan UniversityInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan UniversityInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan UniversityCAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of SciencesInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan UniversityInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan UniversityAbstract Filamentous fungi represent an invaluable source of pharmaceutically active compounds. The development of versatile methods to genetically manipulate filamentous fungi is of great value for improving the low yields of bioactive metabolites and expanding chemical diversity. The CRISPR-Cas9-based system has become a common platform for genome editing in a variety of organisms. However, recent application of this technology in filamentous fungi is limited to model strains, a versatile method for efficient gene disruption in different fungi is lacking. Here, we investigated the utility of the CRISPR-Cas9 system in a less-studied fungus Nodulisporium sp. (No. 65-12-7-1), and we have developed an efficient CRISPR-Cas9-based gene disruption strategy by simultaneous transformation of in vitro transcriptional gRNA and the linear maker gene cassette into the Cas9-expressing fungi. We found that the linear marker gene cassette could not only allow for selection of transformants, but also significantly enhance the gene disruption efficiency by inserting itself into the Cas9 cut site. Moreover, the above approach also demonstrated its efficiency in two other phylogenetically distinct strains Aspergillus oryzae NSAR1 and Sporormiella minima (No. 40-1-4-1) from two different classes of Ascomycota. These results suggested that a versatile CRISPR-Cas9-based gene disruption method in filamentous fungi was established.https://doi.org/10.1038/s41598-017-10052-3 |
| spellingShingle | Yan-Mei Zheng Fu-Long Lin Hao Gao Gen Zou Jiang-Wei Zhang Gao-Qian Wang Guo-Dong Chen Zhi-Hua Zhou Xin-Sheng Yao Dan Hu Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology Scientific Reports |
| title | Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology |
| title_full | Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology |
| title_fullStr | Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology |
| title_full_unstemmed | Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology |
| title_short | Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology |
| title_sort | development of a versatile and conventional technique for gene disruption in filamentous fungi based on crispr cas9 technology |
| url | https://doi.org/10.1038/s41598-017-10052-3 |
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