Efficient CRISPR/Cas9 system of Cordyceps militaris and application in breeding
Qing Liu, Guoliang Meng, Miao Wang, Xiao Li, Mengqian Liu, Fen Wang, Ying Yangand Caihong Dong *
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China
Cordyceps militaris is a popular edible fungus with important economic value worldwide. In this study, an efficient CRISPR/Cas9 genome-editing system based on an autonomously replicating plasmid with an AMA1 sequence was constructed and used in breeding. A precisely targeted gene deletion via homology-directed repair was effectively introduced in C. militaris. Gene editing was successful, with efficiencies of 55.1% and 89% for Cmwc-1 and Cmvvd, respectively. Precisely targeted gene deletion was achieved at an efficiency of 73.9% by a single guide RNA supplementation with donor DNAs. Plasmid loss was observed under non-selective culture conditions, which could permit the recycling of the selectable marker and avoid the adverse effects of the CRISPR/Cas9 system on the fungus, which is beneficial for the generation of new cultivars.
The first genomic safe-harbor site, CmSH1 locus was identified in the genome of C. militaris for gene overexpression and complementation. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid with an AMA1 sequence was designed to facilitate alien gene integration into the CmSH1 locus. The marker-free Cmhyd1 overexpression strain by this safe-harbor-targeted CRISPR/Cas9 system showed enhancement of the disease resistance with negligible effect on the agronomic traits of C. militaris. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing.
Funding: This work was supported by grants from National Natural Science Foundation of China (grant numbers 32202569 and 31872163).