Abstract: High- efficiency gene editing system CRISPR / Cas9 system was employed to construct a Flammulina velutipes Mads- 8 gene knockout vector and transformation system. Based on the transcriptome data,efficient targeted point sequence was selected by analyzing the sequence information of gene Mads- 8 to construct transition vecter sgRNA- T with hph as selection marker gene. The target sequence of vecter sgRNA- T was comfirmed by colony PCR and DNA sequencing.Recombinant vector pg Fvs- Cas9- gRNA was constructed using expression vector pg Fvs- cas9 as the frame and verified by PCR methods and digestion with restriction enzyme.Recombinant vector infected Flammulina velutipes protoplast with PEG mediating.Hygromycin resistance plate was used to screen the transformants,and the Cas9 gene was verified by PCR amplifying using genomic DNA as template. Results showed that targeted point sequence was successfully inserted into the recombinant vector followed by the transformation into the genome of Flammulina velutipes. In this work,a complete method of Flammulina velutipes genome knockout and transformation was established. This method will provide theoretical foundation for the following gene functional identification and breeding research in Flammulina velutipes.