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Genome Editing in the Edible Fungus Poria cocos Using CRISPR-Cas9 System Integrating Genome-wide Off-target Prediction and Detection

Summary

This research developed a new genetic engineering tool for modifying the DNA of Poria cocos, an important medicinal mushroom. This breakthrough allows scientists to precisely edit genes in this fungus for the first time, which could lead to improved cultivation and medicinal properties. Impact on everyday life: – Could lead to more effective and affordable medicinal mushroom products – May help develop improved strains with enhanced health benefits – Could increase production efficiency and availability of this traditional medicine – May enable development of new therapeutic applications – Could help preserve and improve this valuable medicinal resource

Background

Poria cocos is an important edible and medicinal fungus with a long history of use in Asia. Its sclerotia are used medicinally and it demonstrates various pharmacological properties including antitumor, immunomodulatory, and anti-inflammatory effects. However, the lack of adequate genetic tools has hindered molecular genetic research and genetic modification of this species.

Objective

To develop and validate a CRISPR-Cas9 genome editing system for Poria cocos by identifying endogenous U6 promoters, constructing sgRNA expression vectors, and establishing protocols for protoplast transformation while integrating genome-wide off-target prediction and detection.

Results

The CRISPR-Cas9 system successfully disrupted the target ura3 gene through mutation, with a targeting editing efficiency of 1.92%. The mutation was a base transformation changing C to G or A to T of Ura3-sgRNA2 target, resulting in missense mutation. One off-target site was detected among the predicted potential off-target locations.

Conclusion

This study represents the first successful implementation of CRISPR-Cas9 genome editing in P. cocos with integrated off-target analysis. The developed system opens new avenues for genetic breeding and commercial production of this valuable edible and medicinal fungus.
  • Published in:Frontiers in Microbiology,
  • Study Type:Laboratory Research,
  • Source: 10.3389/fmicb.2022.966231
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