Trans-nuclei CRISPR/Cas9 For Safe Genome Editing Technology In Mushrooms

Trans-nuclei CRISPR/Cas9 for safe genome editing technology in mushrooms

Daishiro KOSHI1, Junko SUGANO1, Moriyuki KAWAUCHI1, Takehito NAKAZAWA1, Masahiro SAKAMOTO1, Minji OH2, and Yoichi HONDA1*

1Graduate School of Agriculture, Kyoto University, Kitashirakawaoiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan

2National Institute of Horticultural and Herbal Science, Rural Development Administration, 92, Bisan-ro, Soi-myeon, Eumseong-gun, Chungcheongbuk-do 22709, Republic of Korea

*Email: honda.yoichi.5n@kyoto-u.ac.jp

CRISPR/Cas9 is a promising system for genome editing in many organisms including edible mushrooms. It can be applied for the breeding of new strains with desirable functions in industry. We have successfully applied this technology for the first time to a popular edible mushroom, Pleurotus ostreatus. And less spore dikaryotic mutants were successfully isolated by this technology. However, chromosomal integration of the introduced plasmid was frequently observed that hamper safe and efficient non-GM (non-genetically modified) genome editing using plasmid DNA. 

In this study, we developed a new protocol for safe genome editing by focusing on dikaryon state, in which the two nuclei are not fused and exist independently after cross of two compatible monokaryons. P. ostreatus donor monokaryons harboring an introduced expression cassette of Cas9 and gRNA　(gRNA1 or gRNA2)　targeting pyrG, of which disruption confers 5-fluoroorotidine (5-FOA) resistance, was crossed with a compatible recipient monokaryon #61. These dikaryon strains were selected on media with 5-FOA and resistant strains were obtained with high efficiency depending on the gRNA.Small indel was confirmed at the recipient-, as well as donor-, derived target site by genomic PCR. In addition, recipient-derived monokaryon strains were successfully isolated through protoplast-regeneration. These strains were confirmed by genomic PCR to be free of Cas9 fragments. These results suggest that non-GM genome editing across nuclei in the dikaryon state is possible.  The similar method could be applied in many other basidiomycetes fungi as well.

This study was financially supported by NIHHS, Rural Development Administration, Republic of Korea (PJ015554).