영지CRISPR/Cas9 기반 유전자 교정을 위한 RNP/Nanoparticle 시스템 최적화
Optimization of RNP/Nanoparticle Systems for Enhanced CRISPR/ Cas9-Based Gene Editing in Ganoderma lucidum
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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.
목차
ABSTRACT 서론 재료 및 방법 균주 및 배양 원형질체 분리 gRNA의 디자인과 RNP 생산 RNP/나노파티클 복합체 형성 최적화 원형질체, RNP/나노파티클 혼합비 최적화 결과 및 고찰 영지 RNP/나노파티클 복합체 유전자 교정 최적화 RNP/나노파티클 복합체를 활용한 영지 유전자 교정 적요 감사의 글 REFERENCES