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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Univ Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USA
Univ Iowa, Coll Pharm, Dept Chem & Biochem Engn, Iowa City, IA 52242 USAUniv Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USA
Givens, Brittany E.
Naguib, Youssef W.
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Univ Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USA
Minia Univ, Fac Pharm, Dept Pharmaceut, Al Minya 61519, EgyptUniv Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USA
Naguib, Youssef W.
Geary, Sean M.
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Univ Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USAUniv Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USA
Geary, Sean M.
Devor, Eric J.
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Univ Iowa, Dept Obstet & Gynecol, Carver Coll Med, Iowa City, IA 52242 USAUniv Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USA
Devor, Eric J.
Salem, Aliasger K.
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Univ Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USA
Univ Iowa, Coll Pharm, Dept Chem & Biochem Engn, Iowa City, IA 52242 USAUniv Iowa, Coll Pharm, Div Pharmaceut & Translat Therapeut, Iowa City, IA 52242 USA
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Univ British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
BC Childrens Hosp Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC V5Z 4H4, CanadaUniv British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
Kazemian, Pardis
Yu, Si-Yue
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Univ British Columbia, Fac Pharmaceut Sci, Vancouver, BC V6T 1Z3, CanadaUniv British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
Yu, Si-Yue
Thomson, Sarah B.
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Univ British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
BC Childrens Hosp Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC V5Z 4H4, CanadaUniv British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
Thomson, Sarah B.
Birkenshaw, Alexandra
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Univ British Columbia, Fac Pharmaceut Sci, Vancouver, BC V6T 1Z3, CanadaUniv British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
Birkenshaw, Alexandra
Leavitt, Blair R.
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Univ British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
BC Childrens Hosp Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC V5Z 4H4, CanadaUniv British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
Leavitt, Blair R.
Ross, Colin J. D.
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Univ British Columbia, Fac Pharmaceut Sci, Vancouver, BC V6T 1Z3, CanadaUniv British Columbia, Fac Med, Dept Med Genet, Vancouver, BC V6T 1Z3, Canada
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East China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China
Firmenich Aromat China, Shanghai 201108, Peoples R ChinaEast China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China
Yang, Fei
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Xu, Shuqian
Huang, Chao
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Firmenich Aromat China, Shanghai 201108, Peoples R ChinaEast China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China
Huang, Chao
Shao, Zhiqiang
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Firmenich Aromat China, Shanghai 201108, Peoples R ChinaEast China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China
Shao, Zhiqiang
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Hu, Yuefen
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Yang, Yang
Lai, Yongrong
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Guangxi Med Univ, Dept Gastroenterol, Affiliated Hosp 1, Nanning, Guangxi, Peoples R ChinaEast China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China
Lai, Yongrong
Ke, Qing
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Zhejiang Univ, Sch Med, Affiliated Hosp 1, Dept Neurol, Hangzhou 310003, Zhejiang, Peoples R ChinaEast China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China
Ke, Qing
Liang, Dan
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Anhui Med Univ, Dept Obstet & Gynecol, Affiliated Hosp 1, Hefei 230022, Peoples R China
Anhui Med Univ, NHC Key Lab Study Abnormal Gametes & Reprod Tract, Hefei 230032, Anhui, Peoples R ChinaEast China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China
Liang, Dan
Wu, Yuxuan
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East China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China
Firmenich Aromat China, Shanghai 201108, Peoples R ChinaEast China Normal Univ, Shanghai Key Lab Regulatory Biol, Inst Biomed Sci & Sch Life Sci, Shanghai 200241, Peoples R China