CRISPR-Cas9 gene disruption experiments targeting selected genes in a mushroom-producing fungus.

The overall goal of this project was to develop CRISPR-Cas9 technology for gene editing purposes in two different species of mushroom-producing fungi. Specifically, we initially wanted to use CRISPR to shed light on the function of two genes thought to be involved in mushroom production and sexual development in these fungi. After considerable effort, we could not demonstrate bona fide CRISPR activity disrupting either of our two initial fungal gene targets. A recent study has found that the insertion of a short non-coding intron sequence within the transcribed region of Cas9 allowed for robust expression of the protein product of this bacterial gene in the fungal genome. With this in mind, we re-engineered our Cas9 expression construct for use in new gene editing studies in a more user-friendly fungus to demonstrate CRISPR proof-of principle. The gene Ura1 encodes an essential enzyme in an important biosynthetic pathway in fungi. Ura1 was selected as a CRISPR gene target in these new studies, as disruptants of this gene are easy to identify by screening with the chemical 5-FOA. The results of these experiments will be discussed during this presentation.