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

被引:17
|
作者
Liu, Xiaotian [1 ]
Dong, Jianghan [1 ]
Liao, Jian [1 ]
Tian, Li [1 ]
Qiu, Hao [1 ]
Wu, Tao [1 ]
Ge, Feng [1 ]
Zhu, Jing [1 ]
Shi, Liang [1 ]
Jiang, Ailiang [1 ]
Yu, Hanshou [1 ]
Zhao, Mingwen [1 ]
Ren, Ang [1 ,2 ,3 ]
机构
[1] Nanjing Agr Univ, Coll Life Sci, Dept Microbiol, Key Lab Microbiol Engn Agr Environm,Minist Agr, Nanjing 210095, Peoples R China
[2] Nanjing Agr Univ, Sanya Inst, Sanya 572025, Peoples R China
[3] Guizhou Acad Sci, Inst Biol, Guiyang 550009, Peoples R China
基金
中国国家自然科学基金;
关键词
Flammulina filiformis; CRISPR; Cas9; gene editing; dual sgRNAs; U6; promoter; pyrG; MUTAGENESIS; DELETIONS; MUSHROOM;
D O I
10.3390/jof8070693
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
引用
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页数:10
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