The present study aims to disable the gene Ust1 in the edible basidiomycete fungus Pleurotus in order to elucidate the possible function of this gene concerning the growth of this fungus. Previous research on Ust1 and other similar genes in ascomycete fungi have implicated the Ust1 gene product as a critical transcriptional regulator involved processes of sporulation, mating, and dimorphic growth. Specifically, these proteins are major players in the transition to the filamentous stages of dimorphic growth that often precedes fruitification and sporulation. CRISPR is a gene-editing technique utilizing the endonuclease Cas9 that makes a double-stranded break in DNA 3-4 nucleotides upstream of a recognition site termed the PAM sequence. When complexed with a guide RNA sequence that is complementary to a designated sequence in the genome, Cas9 becomes a highly specific gene “knockout” tool by virtue of triggering an error-prone DNA repair process of non-homologous end-joining (NHEJ) at the site of the double-stranded break. Bioinformatics tools were utilized to identify three different Ust1-specific gRNA sequences for use in our in vivo expression experiments. These gRNAs were then inserted via Gibson Assembly into an 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. Based on the restricted growth patterns observed in the Ust1 CRISPR transformants, we tentatively conclude that Ust1 could be a very important gene necessary for normal growth in the fungus Pleurotus ostreatus.
Primary Speaker
Faculty Sponsors