Genome editing in the edible fungus Poria cocos using CRISPR-Cas9 system integrating genome-wide off-target prediction and detection

• DOI: 10.3389/fmicb.2022.966231
• Publication Date: 2022-08-22T06:34:01Z
• Authors: Zhenni Xie; Can Zhong; Xiaoliu Liu; Ziling Wang; Rongrong Zhou; Jing Xie; Shuihan Zhang; Jian Jin
• Keywords: URA3; Guide RNA; Subgenomic mRNA

Poria cocos is an important edible and medicinal fungus with a long history. However, the lack of adequate genetic tools has hindered molecular research modification this species. In study, endogenous U6 promoters were identified by mining data from P. genome, promoter sequence was used to construct sgRNA expression vector pFC332-PcU6. Then, protoplast isolation protocol developed, sgRNA-Cas9 successfully transformed into cells via PEG/CaCl2-mediated transformation approach. Off-target sites genome-widely predicted detected. As result, target marker gene ura3 disrupted CRISPR-Cas9 system. This first report genome editing in using system integrating genome-wide off-target prediction detection. These will open up new avenues for investigation breeding commercial production fungus.