Trans-nuclei CRISPR/Cas9: safe approach for genome editing in the edible mushroom excluding foreign DNA sequences

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.