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ISSN : 1738-0294(Print)
ISSN : 2288-8853(Online)
Journal of Mushrooms Vol.22 No.4 pp.231-235
DOI : http://dx.doi.org/10.14480/JM.2024.22.4.231

Optimization of RNP/Nanoparticle Systems for Enhanced CRISPR/ Cas9-Based Gene Editing in Ganoderma lucidum

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

Despite their historical use, studies on the genetic functions of mushrooms and varietal improvement via biomolecular techniques are limited compared to other organisms. Recent advancements in CRISPR/Cas9 have enabled precise genetic modifications in mushrooms, with RNP-based systems offering high editing efficiency without foreign gene insertion. In this study, we optimized gene-editing conditions for Ganoderma lucidum (Yongji 2) by utilizing RNP/nanoparticle complexes to enhance efficiency. The optimal conditions included a 0.2 M sorbitol buffer (pH 7.0) and a protoplast-to-complex ratio of 10:1. Among eight gRNAs designed for the catA gene, three were identified with high activity, and PEG-mediated transformation resulted in successful gene edits, primarily involving 1 bp deletions. The editing efficiency reached 7–8%, demonstrating that nanoparticle-supported RNP systems are effective for marker-free gene editing in mushrooms. These findings highlight a promising approach for advancing genetic research and varietal improvement in G. lucidum and other mushroom species.

영지CRISPR/Cas9 기반 유전자 교정을 위한 RNP/Nanoparticle 시스템 최적화

초록

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