Publisher：FEMS Microbiology Letters  

First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ΔpyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ΔpyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ΔpyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment.

DOI： 10.1093/femsle/fnad042

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Wood Science  

In a previous study, we reported a transient transformation system using repeated screening for hygromycin B (Hyg) resistance in the basidiomycete Ceriporiopsis subvermispora. In the present study, by combining this technique with CRISPR/Cas9, we demonstrated successful marker-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms as well as a model white-rot fungus. At first, transformant selection mediated by the transient expression of marker genes was demonstrated using a plasmid harboring the Hyg resistance gene (hph) in P. ostreatus. Then, genome editing of fcy1, which confers 5-fluorocytosine (5-FC) resistance to the host cell, was performed by the transient expression of Cas9, gRNA, and hph and strains with 5-FC resistance and Hyg sensitivity were isolated. Additionally, genome editing of fcy1 in these strains was confirmed by Sanger sequencing. To our knowledge, this is the first report of marker-free genome editing through the transient expression of Cas9, gRNA, and hph in agaricomycetes, which opens the door for repeated genome editing in these fungi.

DOI： 10.1186/s10086-022-02033-6

Scopus

Other Link： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85129867285&origin=inward

Language：English   Publishing type：Research paper (scientific journal)   Participation form：Joint(The vice charge)  

DOI： 10.47371/mycosci.2021.05.002

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台  

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台  

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台  

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台  

Language：Japanese   Presentation type：Oral presentation (general)