One important application of the CRISPR method of gene editing is to discern the function of a specific gene. Specifically, one can use CRISPR to “knockout” a target gene, and then infer function from the observed resulting phenotype. CRISPR utilizes a two-component system, consisting of an expressed endonuclease called Cas9, and a gene-specific guide RNA (gRNA) to which is complexed. The overall goal of this project is to utilize the CRISPR/Cas9 gene editing technology to discern the role of the Tea1 gene in the mushroom producing fungi Pleurotus ostreatus. The protein product of Tea1 has been identified as a key transcription factor regulating growth and differentiation in the related fungus Schizophyllum commune. Our hypothesis is that a CRISPR-generated Tea1 “knockout” in Pleurotus will show similar defects to that observed in S. commune. Bioinformatics tools were utilized to identify four different Tea1-specific gRNA sequences for use in our in vivo expression experiments. These gRNAs were then inserted via Gibson Assembly into a RNA Polymerase type III vector designed to express in Pleurotus small nuclear RNAs such as guide RNAs. The resulting recombinant DNA constructs were verified by DNA sequencing, and then co-transformed along with Cas9 into suitable haploid Pleurotus recipients. These latter experiments are currently in progress, the results of which will be discussed in this presentation.