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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. Further, 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. Double genes,  Cmwc‐1  and  Cmvvd , were edited simultaneously with an efficiency of 10%. Plasmid loss was observed under non‐selective culture conditions, which could permit 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. RNA Pol III promoters, endogenous  tRNA   Pro   of  C. militaris,  and chimeric  AfU6‐tRNA   Gly   can be used to improve the efficiency. Polyethylene glycol‐mediated protoplast transformation was markedly more efficient than  Agrobacterium tumefaciens ‐mediated transformation of  C. militaris . To our knowledge, this is the first description of genome editing and precisely targeted gene deletion in mushrooms based on AMA1 plasmids. Our findings will enable the modification of multiple genes in both functional genomics research and strain breeding.

microbial biotechnology

Efficient  CRISPR /Cas9 system based on autonomously replicating plasmid with an  AMA1  sequence and precisely targeted gene deletion in the edible fungus,  Cordyceps militaris