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

Ke Liu,Jun‐Wei Xu,Xuya Yu,Jun‐Liang Tu,Peng Zhao,Hao You,Bin Sun

doi:10.1111/1751-7915.13534

Abstract

Ganoderma lucidum is an important medicinal mushroom in traditional Chinese medicine. However, the lack of adequate genetic tools has hindered molecular genetic research in and the genetic modification of this species. Here, we report that the presence of an intron is necessary for the efficient expression of the heterologous phosphinothricin‐resistance and green fluorescent protein genes in G. lucidum. Moreover, we improved the CRISPR/Cas9‐mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene. Our results showed that the disruption frequency of the orotidine 5’‐monophosphate decarboxylase gene (ura3) in transformants containing the glyceraldehyde‐3‐phosphate dehydrogenase gene intron in the Cas9 plasmid is 14–18 in 107 protoplasts, which is 10.6 times higher than that in transformants without any intron sequence. Furthermore, genomic fragment deletions in the ura3 and GL17624 genes were achieved via a dual sgRNA‐directed CRISPR/Cas9 system in G. lucidum. We achieved a ura3 deletion frequency of 36.7% in G. lucidum. The developed method provides a powerful platform to generate gene deletion mutants and will facilitate functional genomic studies in G. lucidum.

Highlights

Medicinal mushrooms are rich sources of pharmacologically active compounds (Chaturvedi et al, 2018).Ganoderma lucidum, a traditional medicinal mushroom, has been used to improve health and longevity in China for several millennia (Hsu and Cheng, 2018)
These results indicated that the extra intron at the 50 end of the phosphinothricin-resistance gene is required for its efficient expression in G. lucidum
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used in the higher fungus Ganoderma for gene disruption (Qin et al, 2017), its low disruption frequency hampered its application to genome engineering

Summary

Introduction

Medicinal mushrooms are rich sources of pharmacologically active compounds (Chaturvedi et al, 2018). A traditional medicinal mushroom, has been used to improve health and longevity in China for several millennia (Hsu and Cheng, 2018). NHEJ-mediated gene disruption using the CRISPR/Cas system typically produces small insertions and deletions in the target genes. This method usually lacks the ability to disrupt the function of regulatory sequences or elements in the non-coding genome, whereas gene deletion is an effective alternative for achieving these aims (Cai et al, 2018). Dual sgRNA-directed gene deletion using the CRISPR/Cas system has been demonstrated in some species, such as humans, rabbits, Caenorhabditis elegans and Arabidopsis (Chen et al, 2014; Zheng et al, 2014; Song et al, 2016; Durr et al, 2018). This technology provides a useful platform in basic and applied research for gene deletion in medicinal mushrooms

Results

14 Æ 2 17 Æ 1 18 Æ 3

Discussion

Experimental procedures

Biotech 8