Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus

Area of Science:

• Mycology
• Molecular Biology
• Biotechnology

Background:

• Pleurotus ostreatus is a globally significant edible mushroom.
• Classical breeding for trait improvement is laborious and time-consuming.

Purpose of the Study:

• To establish an efficient gene mutagenesis method for non-genetically modified (non-GM) Pleurotus ostreatus.
• To demonstrate CRISPR/Cas9-assisted genome editing in P. ostreatus.

Main Methods:

• Plasmid-based CRISPR/Cas9 system delivery into P. ostreatus protoplasts.
• Targeting of fcy1 and pyrG genes for drug resistance selection.
• Genomic PCR and sequencing for mutation analysis.

Main Results:

• Generated drug-resistant strains of P. ostreatus.
• Confirmed gene editing via small insertions/deletions or fragment insertion at target sites.
• Successfully introduced a mutation in fcy1 using homology-directed repair.

Conclusions:

• CRISPR/Cas9 is an efficient tool for genome editing in P. ostreatus.
• This technology can facilitate molecular breeding of non-GM cultivated mushroom strains.
• The system is applicable for precise gene disruption and heterologous gene insertion.