English
CRISPR/Cas9 RNP-Mediated Disruption of the pyrG Gene in Hypsizygus marmoreus
Beomgyu Park, Hyeon-Su Ro*
Department of Bio & Medical Big Data and Research Institute of Life Sciences, Gyeongsang National University, Republic of Korea
*Email: rohyeon@gnu.ac.kr
Hypsizygus marmoreus is a commercially important mushroom species in East Asia is characterized by dense texture, mild flavor. Due to its long shelf life, it provides advantage in post-harvest handling and distribution over many cultivated mushrooms. Also, H. marmoreus has been reported to contain bioactive compounds such as ergothioneine with antioxidant activity, highlighting its value as a functional food and a promising target for molecular breeding. However, efficient genome editing tools for this species remain limited. In this study, we developed a CRISPR/Cas9 ribonucleoprotein (RNP)-based gene disruption system targeting the pyrG gene in H. marmoreus. The Cas9–gRNA complex was introduced into protoplasts via PEG-mediated transformation to induce targeted DNA cleavage. The pyrG-deficient strains were selected on media containing 5-fluoroorotic acid (5-FOA) and showed a distinctive growth rate those of the wild type on both 5-FOA and minimal media. Sequence analysis confirmed a 16 bp deletion within the exon region that lead to frameshift. The resulting pyrG-deficient strain can serve as a stable host for further transformation by reintroduction of the pyrG gene. Overall, this study establishes a genome editing platform and a reusable selection system for H. marmoreus, providing a foundation for further functional genomics and strain improvement.
