Dual sgRNA‐directed gene deletion in basidiomycete <i>Ganoderma lucidum</i> using the CRISPR/Cas9 system

Liu, Ke; Sun, Bin; You, Hao; Tu, Jun-Liang; Yu, Xuya; Zhao, Peng; Xu, Junwei

doi:10.1111/1751-7915.13534

Cited by 52 publications

(49 citation statements)

References 55 publications

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“…Although the pharmacological effects have been demonstrated, the genes and metabolic pathways involved in the crucial stages of sporulation in G. lucidum are still unknown. With the completion of genome sequencing of G. lucidum [ 7 ] and the development of genetic tools, including gene silencing systems [ 8 ] and dual sgRNA-directed gene deletion systems [ 9 ], molecular genetics research in this species is increasing exponentially [ 10 , 11 , 12 , 13 ]. Since forward genetic dissection of favorable traits is a time-consuming and arduous task in basidiomycete mushrooms, comprehensive analysis using omics technologies has become a feasible option.…”

Section: Introductionmentioning

confidence: 99%

Transcriptional Dynamics of Genes Purportedly Involved in the Control of Meiosis, Carbohydrate, and Secondary Metabolism during Sporulation in Ganoderma lucidum

Cai

Liang

Liu

et al. 2021

Genes

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Ganoderma lucidum spores (GLS), the mature germ cells ejected from the abaxial side of the pileus, have diverse pharmacological effects. However, the genetic regulation of sporulation in this fungus remains unknown. Here, samples corresponding to the abaxial side of the pileus were collected from strain YW-1 at three sequential developmental stages and were then subjected to a transcriptome assay. We identified 1598 differentially expressed genes (DEGs) and found that the genes related to carbohydrate metabolism were strongly expressed during spore morphogenesis. In particular, genes involved in trehalose and malate synthesis were upregulated, implying the accumulation of specific carbohydrates in mature G. lucidum spores. Furthermore, the expression of genes involved in triterpenoid and ergosterol biosynthesis was high in the young fruiting body but gradually decreased with sporulation. Finally, spore development-related regulatory pathways were explored by analyzing the DNA binding motifs of 24 transcription factors that are considered to participate in the control of sporulation. Our results provide a dataset of dynamic gene expression during sporulation in G. lucidum. They also shed light on genes potentially involved in transcriptional regulation of the meiotic process, metabolism pathways in energy provision, and ganoderic acids and ergosterol biosynthesis.

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Section: Introductionmentioning

confidence: 99%

Transcriptional Dynamics of Genes Purportedly Involved in the Control of Meiosis, Carbohydrate, and Secondary Metabolism during Sporulation in Ganoderma lucidum

Cai

Liang

Liu

et al. 2021

Genes

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“…Molecular genetic approaches tools such as genetic transformation, disruption, and deletion of target genes have been developed in G. lucidum [ 12 – 16 ]. However, efficient expression of heterologous genes remains a main challenge in Ganoderma , although it would be valuable for metabolic regulation and molecular breeding.…”

Section: Introductionmentioning

confidence: 99%

Efficient expression of heterologous genes by the introduction of the endogenous glyceraldehyde-3-phosphate dehydrogenase gene intron 1 in Ganoderma lucidum

You

Sun

et al. 2021

Microb Cell Fact

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Background Ganoderma lucidum, a well-known medicinal mushroom, has received wide attention as a promising cell factory for producing bioactive compounds. However, efficient expression of heterologous genes remains a major challenge in Ganoderma, hindering metabolic regulation research and molecular breeding of this species. Results We show that the presence of glyceraldehyde-3-phosphate dehydrogenase gene (gpd) intron 1 at the 5′ end of, the 3′ end of, or within the heterologous phosphinothricin-resistant gene (bar) is efficient for its expression in G. lucidum. The enhanced expression of bar is exhibited by the higher accumulation of mRNA and increased amounts of protein. Moreover, the insertion of the gpd intron 1 in the β-glucuronidase gene (gus) elevates its mRNA accumulation and enzyme activity, which facilitates the use of this reporter gene in Ganoderma. Conclusions This study has demonstrated the importance of the introduction of gpd intron 1 for the efficient expression of bar and gus in G. lucidum. The presence of the gpd intron 1 in heterologous genes increases levels of mRNA accumulation and protein expression in basidiomycete Ganoderma. The developed method may be utilized in upregulating the expression of other heterologous genes in Ganoderma.

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“…Using this genome editing platform, CYP5150L8 and two CRZ genes were disrupted to reveal their function in GA biosynthetic pathway (Li and Zhong, 2020;Wang et al, 2020). Liu et al (2020) improved the CRISPR/Cas9-mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene and achieved the genomic fragment deletions via a dual sgRNA-directed CRISPR/ Cas9 system in G. lucidum. So the detail regulatory mechanism of the key DEGs which obtained from this study needs to be further investigated by genetic editing.…”

Section: Discussionmentioning

confidence: 99%

Transcriptome dynamics and metabolite analysis revealed the candidate genes and regulatory mechanism of ganoderic acid biosynthesis during liquid superficial‐static culture of Ganoderma lucidum

Wang

Zhao

et al. 2020

Microbial Biotechnology

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Ganoderic acid (GA), an important secondary metabolite of Ganoderma lucidum, exhibited many significant pharmacological activities. In this study, the biosynthetic mechanism of GAs was investigated by comparing metabolites and transcriptome dynamics during liquid superficial-static culture (LSSC) and submerged culture (SC). LSSC was a better method to produce GA because thirteen GAs were identified from mycelia by UPLC-QTOF-MS, and the content of all GAs was higher in LSSC than in SC. Ergosterol was accumulated during the SC process in G. lucidum. Transcriptome dynamics analysis revealed CYP5150L8 was the key gene regulating lanosterol flux into GA biosynthesis. Other sixteen CYP450 genes were significantly higher expressed during the culture time in LSSC and could be potential candidate genes associated with the biosynthesis of different GAs. In addition, six of the ten expressed genes in ergosterol biosynthetic pathway shown upregulated at some time points in SC. These results not only provide a fundamental information of the key genes in ergosterol and GA biosynthetic pathway, but also provide directions for future elucidating the regulatory mechanisms of GAs in G. lucidum and enabling us to promote the development and utilization of LSSC at the industrial level.

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Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Cited by 52 publications

References 55 publications

Transcriptional Dynamics of Genes Purportedly Involved in the Control of Meiosis, Carbohydrate, and Secondary Metabolism during Sporulation in Ganoderma lucidum

Transcriptional Dynamics of Genes Purportedly Involved in the Control of Meiosis, Carbohydrate, and Secondary Metabolism during Sporulation in Ganoderma lucidum

Efficient expression of heterologous genes by the introduction of the endogenous glyceraldehyde-3-phosphate dehydrogenase gene intron 1 in Ganoderma lucidum

Transcriptome dynamics and metabolite analysis revealed the candidate genes and regulatory mechanism of ganoderic acid biosynthesis during liquid superficial‐static culture of Ganoderma lucidum

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