Unlocking the Potential for Genetic Engineering of the Straw-Degrading Mushroom Stropharia rugosoannulata by Constructing a CRISPR/Cas9 Gene Editing System

The artificially cultivated edible mushroom Stropharia rugosoannulata is widely promoted and cultivated in China because of its ability to efficiently decompose agricultural and forestry waste. However, methods for CRISPR/Cas9 genome editing have not yet been established for S. rugosoannulata. In this study, we identified three SrU6 promoters in S. rugosoannulata and constructed the CRISPR/Cas9 expression vector GPiE-SrU6. Moreover, we found that mutant strains were obtained only when the expression of the single guide RNA (sgRNA) was driven by the SrU6-3 promoter. We subsequently employed a tandemly repeated SrU6-tRNA-sgRNA module to knock out two sites within the ura3 gene. The expression vector was introduced into the mycelium via Agrobacterium-mediated transformation (ATMT). Following dual selection with 60 μg/mL hygromycin (Hyg) and 0.2 mg/mL 5-fluoroorotic acid (5-FOA), stable transformants were obtained and subcultured. The mutation efficiency at the targeted ura3 locus was subsequently assessed. The CRISPR/Cas9 system successfully disrupted the target marker gene (ura3), achieving an editing efficiency of 14.9%. In summary, this study reports the first successful establishment of a CRISPR/Cas9 genome editing system in S. rugosoannulata. This study not only meets a future need for genetic manipulation tools for S. rugosoannulata but also provides a robust platform for engineering superior strains for eco-circular agriculture.