Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in .

, previously known as Asian , is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in . Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting under the control of the promoter and FfU6 promoter, respectively, were delivered into protoplasts of Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of was identified, and ultimately several mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in , which broadens the application of this advanced tool in Basidiomycetes.