Construction of a CRISPR/Cas9-Mediated Genome Editing System in Lentinula edodes

Suyun Moon; Jee Young An; Yeon-Jae Choi; Youn-Lee Oh; Hyeon-Su Ro; Hojin Ryu

doi:10.1080/12298093.2021.2006401

Construction of a CRISPR/Cas9-Mediated Genome Editing System in Lentinula edodes

Mycobiology. 2021 Nov 25;49(6):599-603. doi: 10.1080/12298093.2021.2006401. eCollection 2021.

Authors

Suyun Moon¹, Jee Young An², Yeon-Jae Choi³, Youn-Lee Oh⁴, Hyeon-Su Ro³, Hojin Ryu¹

Affiliations

¹ Department of Biological Sciences and Biotechnology, Chungbuk National University, Cheongju, Korea.
² AVOCADO BIO Inc, Seoul, Korea.
³ Department of Bio & Medical Big Data and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Korea.
⁴ Mushroom Science Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea.

Abstract

CRISPR/Cas9 genome editing systems have been established in a broad range of eukaryotic species. Herein, we report the first method for genetic engineering in pyogo (shiitake) mushrooms (Lentinula edodes) using CRISPR/Cas9. For in vivo expression of guide RNAs (gRNAs) targeting the mating-type gene HD1 (LeA1), we identified an endogenous LeU6 promoter in the L. edodes genome. We constructed a plasmid containing the LeU6 and glyceraldehyde-3-phosphate dehydrogenase (LeGPD) promoters to express the Cas9 protein. Among the eight gRNAs we tested, three successfully disrupted the LeA1 locus. Although the CRISPR-Cas9-induced alleles did not affect mating with compatible monokaryotic strains, disruption of the transcription levels of the downstream genes of LeHD1 and LeHD2 was detected. Based on this result, we present the first report of a simple and powerful genetic manipulation tool using the CRISPR/Cas9 toolbox for the scientifically and industrially important edible mushroom, L. edodes.

Keywords: CRISPR/Cas9; HD1; Lentinula edodes; gene editing.

Grants and funding

This work was supported by the AvocadoBio, Inc and Golden Seed Project [Grant No. 213007-05-5-SBH20] from the Ministry of Agriculture, Food and Rural Affairs and the New breeding technologies development Program [Project No. PJ01516501], Rural Development Administration of Republic of Korea.