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Multiplex Gene Precise Editing and Large DNA Fragment Deletion by the CRISPR-Cas9-TRAMA System in Edible Mushroom Cordyceps militaris

Summary

This research developed an advanced gene editing tool for the medicinal mushroom Cordyceps militaris. The new system, called CRISPR-Cas9-TRAMA, allows scientists to precisely modify multiple genes and delete large DNA segments in this valuable mushroom species. This advancement could help improve the production of beneficial compounds and understand how the mushroom functions. Impacts on everyday life: – Could lead to improved production of natural medicines from mushrooms – May help develop more stable and productive mushroom strains for food and medicine – Could enable development of new therapeutic compounds from mushrooms – May lead to more affordable and accessible mushroom-based medicines – Could help advance our understanding of how medicinal mushrooms produce beneficial compounds

Background

Cordyceps militaris is a valuable medicinal mushroom containing bioactive compounds like cordycepin, polysaccharides, carotenoids and ergothioneine that have attracted attention from pharmaceutical and cosmetic industries. However, its development faces obstacles including frequent degeneration, lower ingredient production, and lack of powerful genomic engineering tools.

Objective

To develop an efficient marker-free CRISPR-Cas9-TRAMA genomic editing system for achieving multiplex gene precise editing and large synthetic cluster deletion in C. militaris by excavating endogenous tRNA-processed elements and introducing extrachromosomal plasmids alongside homologous templates.

Results

The Cas9-TRAMA system successfully achieved efficient multiplex gene editing and gene reconstruction via both NHEJ and HDR pathways. The system demonstrated capability for protein modification, promoter strength evaluation, and large DNA fragment (10kb) deletion. The system showed 17.9% efficiency for simultaneous four-site editing and 25.6% efficiency for coding gene reconstruction via HDR pathway.

Conclusion

The Cas9-TRAMA editing system provides a scalable method for excavating valuable metabolic resources from edible mushrooms and engineering cellular pathways to understand complex cell behaviors of mushroom-forming fungi. The system can be efficiently removed after editing without leaving foreign DNA, making it a powerful tool for genomic engineering in C. militaris.
  • Published in:Microbial Biotechnology,
  • Study Type:Laboratory Research,
  • Source: 10.1111/1751-7915.14147
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