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Despite the long history of mushroom use, studies examining the genetic function of mushrooms and the development of new varieties via bio-molecular methods are significantly lacking compared to those examining other organisms. However, owing to recent developments, attempts have been made to use a novel gene-editing technique involving CRISPR/Cas9 technology and genetic scissors in mushroom studies. In particular, research is actively being conducted to utilize ribonucleoprotein particles (RNPs) that can be genetically edited with high efficiency without foreign gene insertion for ease of selection. However, RNPs are too large for Cas9 protein to pass through the cell membrane of the protoplasmic reticulum. Furthermore, guide RNA is unstable and can be easily decomposed, which remarkably affects gene editing efficiency. In this study, nanoparticles were used to mitigate the shortcomings of RNP-based gene editing techniques and to obtain transformants stably. We used Lentinula edodes (shiitake mushroom) Sanjo705-13 monokaryon strain, which has been successfully used in previous genome editing experiments. To identify a suitable osmotic buffer for the isolation of protoplast, 0.6 M and 1.2 M sucrose, mannitol, sorbitol, and KCl were treated, respectively. In addition, with various nanoparticle-forming materials, experiments were conducted to confirm genome editing efficiency via the formation of nanoparticles with calcium phosphate (CaP), which can be bound to Cas9 protein without any additional amino acid modification. RNPs/NP complex was successfully formed and protected nuclease activity with nucleotide sequence specificity.

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저자

• 김민식 [ Minseek Kim | 농촌진흥청 국립원예특작과학원 버섯과 ]
• 류호진 [ Hojin Ryu | 충청북도 청주시 충북대학교 자연대학 ]
• 오민지 [ Min Ji Oh | 농촌진흥청 국립원예특작과학원 버섯과 ]
• 임지훈 [ Ji-Hoon Im | 농촌진흥청 국립원예특작과학원 버섯과 ]
• 이종원 [ Jong-Won Lee | 농촌진흥청 국립원예특작과학원 버섯과 ]
• 오연이 [ Youn-Lee Oh | 농촌진흥청 국립원예특작과학원 버섯과 ] Corresponding author

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