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Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis

Authors :
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
Source :
Journal of Fungi, Vol 8, Iss 7, p 693 (2022)
Publication Year :
2022
Publisher :
MDPI AG, 2022.

Abstract

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.

Details

Language :
English
ISSN :
2309608X
Volume :
8
Issue :
7
Database :
Directory of Open Access Journals
Journal :
Journal of Fungi
Publication Type :
Academic Journal
Accession number :
edsdoj.9369ad2473be498e9686eda8fb93413e
Document Type :
article
Full Text :
https://doi.org/10.3390/jof8070693