Multiplex gene precise editing and large DNA fragment deletion by the CRISPR‐Cas9‐TRAMA system in edible mushroom Cordyceps militaris

The medicinal mushroom Cordyceps militaris contains abundant valuable bioactive ingredients that have attracted a great deal of attention in the pharmaceutical and cosmetic industries. However, the development of this valuable mushroom faces the obstacle of lacking powerful genomic engineering tools. Here, by excavating the endogenous tRNA‐processed element, introducing the extrachromosomal plasmid and alongside with homologous template, we develop a marker‐free CRISPR‐Cas9‐TRAMA genomic editing system to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris. We further operated editing in the synthetases of cordycepin and ergothioneine to demonstrate the application of Cas9‐TRAMA system in protein modification, promoter strength evaluation and 10 kb metabolic synthetic cluster deletion. The Cas9‐TRAMA system provides a scalable method for excavating the valuable metabolic resource of medicinal mushrooms and constructing a mystical cellular pathway to elucidate the complex cell behaviours of the edible mushroom.