Genome Editing in the Edible Fungus Using CRISPR-Cas9 System Integrating Genome-wide Off-target Prediction and Detection

is an important edible and medicinal fungus with a long history. However, the lack of adequate genetic tools has hindered molecular genetic research and the genetic modification of this species. In this study, the endogenous U6 promoters were identified by mining data from the genome, and the promoter sequence was used to construct a sgRNA expression vector pFC332-PcU6. Then, the protoplast isolation protocol was developed, and the sgRNA-Cas9 vector was successfully transformed into the cells of PEG/CaCl2-mediated transformation approach. Off-target sites were genome-widely predicted and detected. As a result, the target marker gene was successfully disrupted by the CRISPR-Cas9 system. This is the first report of genome editing in using CRISPR-Cas9 system integrating genome-wide off-target prediction and detection. These data will open up new avenues for the investigation of genetic breeding and commercial production of edible and medicinal fungus.