| Summary: | <i>Flammulina filiformis</i>, previously known as Asian <i>Flammulina velutipes</i>, 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 <i>F. filiformis</i>. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting <i>pyrG</i> under the control of the <i>gpd</i> promoter and FfU6 promoter, respectively, were delivered into protoplasts of <i>F. filiformis</i> Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of <i>F. filiformis</i> was identified, and ultimately several <i>pyrG</i> 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 <i>pyrG</i> 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 <i>F. filiformis</i>, which broadens the application of this advanced tool in Basidiomycetes.
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