Gene editing using CRISPR/Cas9 is an innovative tool for developing new mushroom strains, offering a promising alternative to traditional breeding methods that are time-consuming and labor-intensive. However, plasmid-based gene editing presents several challenges, including the need for selecting appropriate promoters for Cas9 expression, optimizing codons for the Cas9 gene, the unintended insertion of fragmented plasmid DNA into genomic DNA (gDNA), and regulatory concerns related to genetically modified organisms (GMOs). To address these issues, we utilized a Ribonucleoprotein (RNP) complex consisting of Cas9 and gRNA for gene editing to modify the A mating-type gene of Lentinula edodes. To overcome the challenges posed by the large size of the Cas9 protein, which limits its penetration through the protoplast membrane, and the susceptibility of sgRNA to degradation, we developed a nanoparticle complex using calcium phosphate and polyacrylic acid. This approach significantly improved gene editing efficiency. Consequently, we successfully edited the mating-controlling genes hd1 and hd2 in L. edodes and examined the effects of their disruption on mating. Disruption of the hd1 gene, which is known to influence mycelial growth, did not significantly affect growth or mating. In contrast, editing the hd2 gene disrupted mating with compatible partners, highlighting its critical role in the mating process. The RNP-based transformation technology presented here offers significant advancement over traditional plasmid-based methods, enhancing the efficiency of targeted gene modification while avoiding the insertion of foreign genetic material, thereby mitigating GMO-related regulatory concerns.
Keywords: CRISPR/Cas9; DNA-free; RNP; homeodomain; mating type; nanoparticle.