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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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