Marker-Free Gene Editing Through RNP/Nanoparticle Complex In Lentinul edodes

 Despite the significance of mushrooms in human culture and their close relationship, studies on their genetic information has lagged behind other eukaryotic organisms. This is due to the prevalence of non-homologous end-joining (NHEJ) mechanisms in mushrooms, which makes it challenging to apply homologous recombination (HR) mechanisms commonly used in the genetic studies of eukaryotic cells. However, with the emergence of the third-generation gene-editing tool CRISPR/Cas9, which received the Nobel Prize in 2020, research on genome editing in the field of mushrooms has begun. Particularly, the use of CAS protein and guide RNA alone for ribonucleoprotein (RNP) gene editing has gained momentum, as it eliminates the need for the insertion of foreign genes. In this study, experiments were conducted to enhance the efficiency of marker-free gene editing using RNP by forming nanoparticle complex. The suitability of osmotic buffers for protoplast isolation was confirmed using the monokaryotic strain Sanjo705-13 of Lentinula edodes. The highest protoplast isolation efficiency of 3.61×108/mL was observed in a 0.6M sucrose solution. Subsequently, nanoparticles composed of calcium phosphate(CaP) and polyacrylic acid were generated to form RNP/nanoparticle(RNP/NP) complexes. For this experiment, gRNA gHD1-1 and gHD1-2 were designed targeting HD1 gene of L. edodes. After isolating gene editing strains, analysis revealed the separation of 12 strains with gene editing from gHD1-1 and 7 strains from gHD1-2. Among the isolated strains, cases of multiple editings were also confirmed. 

 This work was supported by grants from the New Breeding Technologies Development Program [Project No. RS-2022-RD010233], Rural Development Administration (RDA), Republic of Korea.