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Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis
Flammulina filiformis, previously known as Asian Flammulina velutipes, 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 t...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9318071/ https://www.ncbi.nlm.nih.gov/pubmed/35887449 http://dx.doi.org/10.3390/jof8070693 |
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author | Liu, Xiaotian Dong, Jianghan Liao, Jian Tian, Li Qiu, Hao Wu, Tao Ge, Feng Zhu, Jing Shi, Liang Jiang, Ailiang Yu, Hanshou Zhao, Mingwen Ren, Ang |
author_facet | Liu, Xiaotian Dong, Jianghan Liao, Jian Tian, Li Qiu, Hao Wu, Tao Ge, Feng Zhu, Jing Shi, Liang Jiang, Ailiang Yu, Hanshou Zhao, Mingwen Ren, Ang |
author_sort | Liu, Xiaotian |
collection | PubMed |
description | Flammulina filiformis, previously known as Asian Flammulina velutipes, 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 F. filiformis. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting pyrG under the control of the gpd promoter and FfU6 promoter, respectively, were delivered into protoplasts of F. filiformis Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of F. filiformis was identified, and ultimately several pyrG 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 pyrG 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 F. filiformis, which broadens the application of this advanced tool in Basidiomycetes. |
format | Online Article Text |
id | pubmed-9318071 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-93180712022-07-27 Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis Liu, Xiaotian Dong, Jianghan Liao, Jian Tian, Li Qiu, Hao Wu, Tao Ge, Feng Zhu, Jing Shi, Liang Jiang, Ailiang Yu, Hanshou Zhao, Mingwen Ren, Ang J Fungi (Basel) Communication Flammulina filiformis, previously known as Asian Flammulina velutipes, 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 F. filiformis. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting pyrG under the control of the gpd promoter and FfU6 promoter, respectively, were delivered into protoplasts of F. filiformis Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of F. filiformis was identified, and ultimately several pyrG 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 pyrG 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 F. filiformis, which broadens the application of this advanced tool in Basidiomycetes. MDPI 2022-06-30 /pmc/articles/PMC9318071/ /pubmed/35887449 http://dx.doi.org/10.3390/jof8070693 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Communication Liu, Xiaotian Dong, Jianghan Liao, Jian Tian, Li Qiu, Hao Wu, Tao Ge, Feng Zhu, Jing Shi, Liang Jiang, Ailiang Yu, Hanshou Zhao, Mingwen Ren, Ang Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis |
title | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis |
title_full | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis |
title_fullStr | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis |
title_full_unstemmed | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis |
title_short | Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis |
title_sort | establishment of crispr/cas9 genome-editing system based on dual sgrnas in flammulina filiformis |
topic | Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9318071/ https://www.ncbi.nlm.nih.gov/pubmed/35887449 http://dx.doi.org/10.3390/jof8070693 |
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