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

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 (<i>Lentinula edodes</i>) using CRISPR/Cas9. For <i>in vivo</i> expression of guide RNAs (gRNAs) targeting the mating-type gene <i>HD1</i> (<i>LeA1</i>), we identified an endogenous <i>LeU6</i> promoter in the <i>L. edodes</i> genome. We constructed a plasmid containing the <i>LeU6</i> and <i>glyceraldehyde-3-phosphate dehydrogenase</i> (<i>LeGPD</i>) promoters to express the Cas9 protein. Among the eight gRNAs we tested, three successfully disrupted the <i>LeA1</i> 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, <i>L. edodes</i>.