Safe-Harbor-Targeted CRISPR/Cas9 System and <i>Cmhyd1</i> Overexpression Enhances Disease Resistance in <i>Cordyceps militaris</i>
Qing Liu, Guoliang Meng, Miao Wang, Xiao Li, Mengqian Liu, Fen Wang, Ying Yang, Caihong Dong
Abstract
Fungal disease of mushroom Cordyceps militaris (CM) caused by Calcarisporium cordycipiticola (CC) is destructive to fruiting body cultivation, resulting in significant economic loss and potential food safety risks. CRISPR/Cas9 genome editing has proven to be a powerful tool for crop improvement but seldom succeeded in mushrooms. Here, the first genomic safe-harbor site, CmSH1 locus, was identified in the CM genome. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid was designed to facilitate alien gene integration at the CmSH1 locus. Cmhyd1, one of the hydrophobin genes, was confirmed as a defensive factor against CC infection, and Cmhyd1 overexpression by this system showed enhancement of disease resistance with negligible effect on the agronomic traits of CM. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing. This study provided the first safe harbor site for genetic manipulations, a safe harbor-targeted CRISPR/Cas9 system, and the first disease-resistant gene-editing breeding system in mushrooms.