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

Xie, Zhenni; Zhong, Can; Liu, Xiaoliu; Wang, Ziling; Zhou, Rongrong; Xie, Jing; Zhang, Shuihan; Jin, Jian

doi:10.3389/fmicb.2022.966231

Cited by 10 publications

(5 citation statements)

References 48 publications

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“…Based on the principle of cost-effectiveness, it is necessary to specify the optimal enzymatic concentration when the maximum protoplast yield is reached. Currently, the commonly used cell wall-degrading enzymes include snailase, driselase and lywallzyme ( Han et al, 2020 ; Ning et al, 2022 ; Xie et al, 2022 ). Snailase is a complex enzyme that consists of over 20 different types of enzymes, e.g., cellulase, pectinase, mannanase, glucoamylase, chitinase and lipase ( Liang et al, 2012 ).…”

Section: Discussionmentioning

confidence: 99%

“…The hygromycin phosphotransferase gene is widely used as an antibiotic transformation marker in fungi, including Eutypella sp. ( Ning et al, 2022 ), Poria cocos ( Xie et al, 2022 ) and Gloeophyllum trabeum ( Li et al, 2023 ). Here, we determined that the lowest hygromycin B inhibition concentration in C. chanhua 022017–3 was 300 μg/mL.…”

Section: Discussionmentioning

confidence: 99%

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Establishment of a genetic transformation system for cordycipitoid fungus Cordyceps chanhua

Cai,

Xiao,

Xing

et al. 2024

Front. Microbiol.

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Cordyceps chanhua is a well-known edible and medicinal mushroom with a long history of use in China, and it contains a variety of secondary metabolites with interesting bioactive ingredients. However, recent researches have mainly focused on cultivation conditions, secondary metabolite compositions and pharmacological activities of C. chanhua, the lack of an efficient and stable genetic transformation system has largely limited further research on the relationship between secondary metabolites and biosynthetic gene clusters in C. chanhua. In this study, single-factor experiments were used to compare the effects of different osmotic stabilizers, enzyme concentrations and enzyme digestion times on protoplast yield, and we found that the highest yield of 5.53 × 108 protoplasts/mL was obtained with 0.7 M mannitol, 6 mg/mL snail enzyme and 4 h of enzyme digestion time, and the regeneration rate of protoplasts was up to approximately 30% using 0.7 M mannitol as an osmotic stabilizer. On this basis, a PEG-mediated genetic transformation system of C. chanhua was successfully established for the first time, which lays the foundation for further genetic transformation of C. chanhua.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Establishment of a genetic transformation system for cordycipitoid fungus Cordyceps chanhua

Cai,

Xiao,

Xing

et al. 2024

Front. Microbiol.

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“…The PEG-mediated protoplast transformation was performed as previously described with minor modifications [ 21 , 33 ]. Protoplasts were digested with 1.5% lysozyme (Guangdong Culture Collection Center, Guangzhou, China) at 80 rpm for 2 h at 28 °C and filtered through repeated washing with 0.6 M mannitol.…”

Section: Methodsmentioning

confidence: 99%

Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing

Liu,

Xie,

Xie

et al. 2024

Microb Cell Fact

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Background Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. Result In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. Conclusion The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.

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“…The PEG-mediated protoplast transformation was performed as previously described with minor modi cations [43,44]. Protoplasts were digested with 1.5% lysozyme (Guangdong Culture Collection Center, Guangzhou, China) at 80 rpm for 2 h at 28°C and ltered through repeated washing with 0.6 M mannitol.…”

Section: Peg-mediated Protoplast Transformationmentioning

confidence: 99%

Functional validation of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing

Liu

Xie

et al. 2023

Preprint

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Background Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. In recent years, CRISPR/Cas9 gene editing technology has emerged and other functional genes in the Poria cocos triterpene synthesis pathway have been studied; PcSE has not been reported. Results In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. sgRNA sequences were designed based on the active site residues. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. Which showed that deletion of PcSE inhibited triterpene synthesis. Conclusions A gene editing system based on molecular docking was successfully constructed to demonstrate that PcSE functions as a house squalene cyclooxygenase, which provides a basis for further studies on the heterologous biosynthesis of P. cocos secondary metabolites.

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

Cited by 10 publications

References 48 publications

Establishment of a genetic transformation system for cordycipitoid fungus Cordyceps chanhua

Establishment of a genetic transformation system for cordycipitoid fungus Cordyceps chanhua

Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing

Functional validation of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing

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