Establishment of Lentinula edodes genome editing technology and analysis of genes related to preservation of fruiting body

2023 Fiscal Year Final Research Report

• Project/Area Number: 21K05700
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 40020:Wood science-related
• Research Institution: Kitami Institute of Technology
• Principal Investigator: Sato Toshitsugu 北見工業大学, 工学部, 教授 (00390881)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: シイタケ / ゲノム編集 / CRISPR/Cas9 / RNP / 胞子 / sgRNA / ドナーベクター

Outline of Final Research Achievements

In Lentinula edodes, genome editing technology by Cas9 protein-gRNA complex (RNP) has not yet been established. In this study, we examined the conditions for spore preservation, and also the design of sgRNAs for the sdc-ip and lcc1 genes and the construction of donor vectors for each gene modification in order to establish genome editing by RNPs. As a result, it was revealed that spores with a germination rate of approximately 3% immediately after collection from fruiting body could maintain a germination rate of 1% for 60 days at -80°C. We also constructed donor vectors to convert the sdc-ip gene in L. edodes to cbx, which confers carboxin resistance by artificial single nucleotide substitution, and to replace the lcc1 gene with cbx to destroy the lcc1 gene.

Free Research Field

農学

Academic Significance and Societal Importance of the Research Achievements

本研究では、シイタケではいまだに確立されていない、外来遺伝子を細胞に残さないCas9タンパク質gRNA複合体（RNP）によるゲノム編集技術を確立することを目的としている。この技術の確立は、遺伝子組換え体とならない実用化可能なシイタケの育種技術となるため、学術的にも社会的にも意義がある。