Koshi Daishiro, Sugano Junko, Yamasaki Fuga, Kawauchi Moriyuki, Nakazawa Takehito, Oh Minji, Honda Yoichi
Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kitashirakawaoiwakecho, Kyoto, 606-8502, Japan.
Mushroom Research Division, Rural Development Administration, National Institute of Horticultural and Herbal Science, Bisanro 92, Eumseong, Chungbuk, 27709, Republic of Korea.
Appl Microbiol Biotechnol. 2024 Dec 30;108(1):548. doi: 10.1007/s00253-024-13367-0.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions. In this study, we developed a foreign-DNA-free genome editing method in Pleurotus ostreatus by transferring the Cas9/guide RNA (gRNA) complex between nuclei in the dikaryotic state. We isolated a donor monokaryotic P. ostreatus strain expressing Cas9 and gRNA targeting pyrG by introducing a recombinant plasmid, which exhibited uracil auxotrophy and 5-fluoroorotic acid (5-FOA) resistance. This strain was then crossed with a pyrG recipient monokaryon, resulting in dikaryotic strains exhibiting 5-FOA resistance after mycelial growth. When these strains were de-dikaryonized into monokaryons through protoplasting, we obtained monokaryotic isolates harboring the recipient nucleus with small indels at the pyrG target site. Importantly, these isolates were confirmed to be free of foreign DNA through genomic PCR, Southern blotting, and whole-genome resequencing analyses. This is the first report of an efficient genome editing protocol in agaricomycetes that ensures no integration of exogenous DNA. This approach is expected to be applicable to other fungi with a dikaryotic life cycle, opening new possibilities for molecular breeding without the concerns associated with GMOs. KEY POINTS: • Successful genome editing via CRISPR/Cas9 trans-nuclei manner in P. ostreatus. • Recipient monokaryons from gene-edited dikaryons showed no exogenous DNA sequences. • Efficient genome editing protocol for safer molecular breeding in mushroom fungus.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）辅助的基因组编辑已应用于几种主要的可食用伞菌纲真菌，实现了高效的基因靶向。该方法对于快速高效地培育高价值品种以及克服栽培挑战具有广阔前景。然而，在此过程中外源DNA片段的整合引发了对转基因生物（GMO）及其监管限制的担忧。在本研究中，我们通过在双核状态的细胞核之间转移Cas9/引导RNA（gRNA）复合物，开发了一种平菇无外源DNA的基因组编辑方法。我们通过引入重组质粒分离出了一个表达针对pyrG的Cas9和gRNA的供体单核平菇菌株，该菌株表现出尿嘧啶营养缺陷型和5-氟乳清酸（5-FOA）抗性。然后将该菌株与pyrG受体单核体杂交，菌丝体生长后得到表现出5-FOA抗性的双核菌株。当这些菌株通过原生质体化去双核化为单核体时，我们获得了在pyrG靶位点带有小插入缺失的含有受体细胞核的单核分离株。重要的是，通过基因组PCR、Southern印迹和全基因组重测序分析证实这些分离株不含外源DNA。这是伞菌纲真菌中首个确保无外源DNA整合的高效基因组编辑方案的报道。这种方法有望应用于其他具有双核生命周期的真菌，为分子育种开辟新的可能性，而无需担心转基因生物相关问题。要点：• 通过CRISPR/Cas9跨核方式在平菇中成功进行基因组编辑。• 基因编辑双核体产生的受体单核体无外源DNA序列。• 用于蘑菇真菌更安全分子育种的高效基因组编辑方案。
