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ISSN : 1738-0294(Print)
ISSN : 2288-8853(Online)
Journal of Mushrooms Vol.23 No.3 pp.178-181
DOI : http://dx.doi.org/10.14480/JM.2025.23.3.178

Report on the instability of CRISPR/Cas9-mediated gene-edited Ganoderma lucidum due to multinucleation

Minseek Kim, Min Ji Oh, Ji-Hoon Im, Eun-Ji Lee, Sung-I Woo, Youn-Lee Oh*
Mushroom Science Division, National Institute of Horticultural and Herbal Science, RDA, Eumseong 27709, Korea

Abstract

CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing has recently been applied to Ganoderma lucidum as a promising tool for functional genomics and strain improvement. However, the multinuclear nature of this basidiomycete can result in genetic mosaicism, raising concerns about the long-term stability of edited strains. In this study, we report the occurrence of revertant phenotypes in CRISPR/Cas9-edited transformants of G. lucidum. Although the edited colonies initially exhibited the expected phenotype, repeated subculturing led to the reappearance of wild-type phenotypes. PCR and sequencing analyses revealed the coexistence of edited and non-edited nuclei, and the progressive loss of edited genotypes over successive generations. These findings demonstrate that multinuclearity is a key factor contributing to the instability of CRISPR/Cas9-based edits in G. lucidum. This brief report provides the first direct documentation of revertant occurrence in edited G. lucidum strains and underscores the need for rigorous selection strategies and novel approaches to secure stable homokaryotic transformants in mushroom genetic engineering.

다핵성에 기인한 영지버섯 CRISPR/Cas9 유전자 교정체의 불안정성

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