Effect of a Mating Type Gene Editing in Lentinula edodes Using RNP/Nanoparticle Complex.

Area of Science:

• Mycology
• Molecular Biology
• Biotechnology

Background:

• CRISPR/Cas9 gene editing offers advantages over traditional mushroom breeding.
• Plasmid-based gene editing faces challenges like promoter selection, codon optimization, DNA insertion, and GMO regulations.

Purpose of the Study:

• To develop an efficient and safe gene editing method for Lentinula edodes.
• To overcome limitations of plasmid-based CRISPR/Cas9 delivery using Ribonucleoprotein (RNP) complexes and nanoparticle technology.

Main Methods:

• Utilized a Ribonucleoprotein (RNP) complex of Cas9 and gRNA for gene editing.
• Developed a calcium phosphate and polyacrylic acid nanoparticle complex for RNP delivery to overcome size and degradation issues.
• Targeted the A mating-type genes (hd1 and hd2) in Lentinula edodes.

Main Results:

• Successfully edited the hd1 and hd2 genes in Lentinula edodes.
• Disruption of hd1 did not significantly impact mycelial growth or mating.
• Editing the hd2 gene disrupted mating, confirming its essential role in the mating process.

Conclusions:

• RNP-based gene editing with nanoparticle delivery is a highly efficient method for Lentinula edodes.
• This approach circumvents the drawbacks of plasmid-based methods, including foreign DNA insertion and GMO concerns.
• The study provides an advanced tool for genetic modification in edible mushrooms.