Using CRIPSR‐Cas9 to Study Repeat Variation in Azf1p

Abstract

Repetitive regions within the genome can code for proteins that have repetitive amino acid sequences. These genomic regions can expand or contract and, in the few cases studied, these changes can affect protein function. This project tests how variation in the repetitive regions of the prion‐forming protein Azf1p, cause changes in gene expression and prion formation. Azf1p has two repetitive regions, a polyQ and a polyN, and is a transcriptional activator. We used CRIPSR‐Cas9 gene editing technology to cut within repetitive regions and allow the inherent DNA repair mechanisms in budding yeast, S. cerevisiae, to fix the break and create expansions or contractions. Subsequently we tested the effect of the repeat variation in different assays, including spotting assays, growth assays, phenotypic assays and qPCR analysis. We generated several variants in the polyQ domain of the AZF1 gene which were confirmed via sequencing. The research presented here will assess how variation within these regions affects various functions of AZF1, including growth on different carbon sources, colony morphology and changes in target gene expression. This work hopes to both create ways of using CRISPR‐Cas9 on repetitive regions that can be useful in the study of the function of these repeats and identify new functional domains within an important transcription factor, AZF1.