Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in <i>Flammulina filiformis</i>
<i>Flammulina filiformis</i>, previously known as Asian <i>Flammulina velutipes</i>, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionar...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-06-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/8/7/693 |
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| author | Xiaotian Liu Jianghan Dong Jian Liao Li Tian Hao Qiu Tao Wu Feng Ge Jing Zhu Liang Shi Ailiang Jiang Hanshou Yu Mingwen Zhao Ang Ren |
| author_facet | Xiaotian Liu Jianghan Dong Jian Liao Li Tian Hao Qiu Tao Wu Feng Ge Jing Zhu Liang Shi Ailiang Jiang Hanshou Yu Mingwen Zhao Ang Ren |
| author_sort | Xiaotian Liu |
| collection | DOAJ |
| description | <i>Flammulina filiformis</i>, previously known as Asian <i>Flammulina velutipes</i>, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in <i>F. filiformis</i>. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting <i>pyrG</i> under the control of the <i>gpd</i> promoter and FfU6 promoter, respectively, were delivered into protoplasts of <i>F. filiformis</i> Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of <i>F. filiformis</i> was identified, and ultimately several <i>pyrG</i> mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these <i>pyrG</i> mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in <i>F. filiformis</i>, which broadens the application of this advanced tool in Basidiomycetes. |
| first_indexed | 2024-03-09T03:18:07Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-09T03:18:07Z |
| publishDate | 2022-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-9369ad2473be498e9686eda8fb93413e2023-12-03T15:15:44ZengMDPI AGJournal of Fungi2309-608X2022-06-018769310.3390/jof8070693Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in <i>Flammulina filiformis</i>Xiaotian Liu0Jianghan Dong1Jian Liao2Li Tian3Hao Qiu4Tao Wu5Feng Ge6Jing Zhu7Liang Shi8Ailiang Jiang9Hanshou Yu10Mingwen Zhao11Ang Ren12Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China<i>Flammulina filiformis</i>, previously known as Asian <i>Flammulina velutipes</i>, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in <i>F. filiformis</i>. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting <i>pyrG</i> under the control of the <i>gpd</i> promoter and FfU6 promoter, respectively, were delivered into protoplasts of <i>F. filiformis</i> Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of <i>F. filiformis</i> was identified, and ultimately several <i>pyrG</i> mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these <i>pyrG</i> mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in <i>F. filiformis</i>, which broadens the application of this advanced tool in Basidiomycetes.https://www.mdpi.com/2309-608X/8/7/693<i>Flammulina filiformis</i>CRISPR/Cas9gene editingdual sgRNAsU6 promoter<i>pyrG</i> |
| spellingShingle | Xiaotian Liu Jianghan Dong Jian Liao Li Tian Hao Qiu Tao Wu Feng Ge Jing Zhu Liang Shi Ailiang Jiang Hanshou Yu Mingwen Zhao Ang Ren Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in <i>Flammulina filiformis</i> Journal of Fungi <i>Flammulina filiformis</i> CRISPR/Cas9 gene editing dual sgRNAs U6 promoter <i>pyrG</i> |
| title | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in <i>Flammulina filiformis</i> |
| title_full | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in <i>Flammulina filiformis</i> |
| title_fullStr | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in <i>Flammulina filiformis</i> |
| title_full_unstemmed | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in <i>Flammulina filiformis</i> |
| title_short | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in <i>Flammulina filiformis</i> |
| title_sort | establishment of crispr cas9 genome editing system based on dual sgrnas in i flammulina filiformis i |
| topic | <i>Flammulina filiformis</i> CRISPR/Cas9 gene editing dual sgRNAs U6 promoter <i>pyrG</i> |
| url | https://www.mdpi.com/2309-608X/8/7/693 |
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