Using CRISPR-Cas9 to Study the Effect of Repeat Variation on Azf1p
Parmenter, Mackenzie L.
2020
- Proteins containing repeat regions are coded for by DNA that is inherently unstable and genetic variation within these regions can alter protein function. This thesis examines a new way of creating these genomic repeat variants for experimental purposes by using CRISPR-Cas9 and allowing error in double stranded break repair mechanisms to create variants. My sequencing data indicates that cutting ... read morewithin a repetitive region leads to more mutagenesis than when cutting outside a repetitive region but providing a homologous template. This novel protocol made the generation of mutants quicker than traditional methods but has limited control and can yield limited outcomes. This system was applied to a protein of interest, Azf1p, that is a prion-forming transcriptional activator that activates different genes under different carbon conditions. AZF1 has two repetitive regions, a polyQ and a polyN and four different genomic repeat length variants were created, three in the polyQ region and one in the polyN region. The effect of polyQ repeat variation on the function of Azf1p was tested. Preliminary data indicates that polyQ variation does not have an effect on growth on different carbon sources or target gene expression. More research must go into studying the effect of repeat variation on the ability to form and propagate the [AZF1+] prion, but I propose that the polyQ region may play a role.read less
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