Date of Award
Master of Science (MS)
School of Biological Sciences
First adopted as a model organism by Shear and Dodge in 1927, the filamentous fungus Neurospora crassa is among the most well studied eukaryotic systems today, facilitating major discoveries in genetics, biochemistry, and molecular biology. The sequencing of the Neurospora genome was completed in 2003, which combined with the organism’s fast growth, simple culturing requirements, and haploid life cycle, make it a particularly attractive model system. Despite these advantages, Neurospora crassa’s research value is diminished by its recalcitrance to expressing ectopic sequences incorporated into its genome. As a result, molecular techniques requiring heterologous expression (such as CRISPR) are largely incompatible with Neurospora research, despite successful implementation in other model systems. Neurospora possesses multiple well-documented processes of neutralizing sequences deemed a threat to genomic integrity, such as transposable elements or mycoviruses. Even in the absence of characterized genome defense mechanisms in N. crassa, such as quelling, the expression of transgenic Cas9 endonuclease remains inhibited, suggesting the possibility of an additional genome defense mechanism operating within this model system. Here I propose that inhibition of Cas9 expression in N. crassa results from regulatory elements, encoded within its genome, that target transgenic sequences for silencing. I further propose that these regulatory elements are, themselves, vulnerable to inactivation through mutation. Utilizing an experimental transgenic strain containing inexpressible Cas9 fragment, mutagenesis assays have been performed and a potential heterologous expression positive (hep) mutant has been isolated. Sequencing of the transgenic construct in the mutant revealed no alterations, indicating that this mutation likely acts in trans. Furthermore, the results of sexual crosses and recombinant screening indicate that a single mutation located in the vicinity of the native leu-1 locus on chromosome three is likely responsible for the observed mutant phenotype.
Mierendorf, James Christopher, "Investigating Heterologous Transgene Expression in N. Crassa" (2023). Theses and Dissertations. 1690.