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Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder causing fluid-filled cysts to develop within in the kidneys . This is believed to occur through a loss of function of the PDK1 gene. In humans, but not mice, evidence has emerged that alternative DNA structure formation is responsible for gene inactivation. In order to better understand genetic alterations at the endogenous PKD1 locus, and preserve DNA structures, a method needs to be utilized that identifies a specific locus but does not denature the genome. We adapted a previously published protocol using a fluorescently tagged dCAS9 protein (Deng et al. in 2015 ) with the goal of identifying specific loci in human and murine cells. Our first aim was to apply the method to visualize telomeric DNA. This method utilized nuclease deficient CRISPR-associated caspase 9 (dCAS9) fused to the halo protein, a fluorescent halo ligand, and single guide RNA (sgRNA) to telomeric repeats . We anticipated visualization of multiple signals within nuclei, corresponding to labeled dCAS9 binding to the telomeres. Following this procedure, only a slight overlap with nuclear DNA was visualized, which implies the need for further procedure adjustments . This method may provide the framework for future genetic studies using fluorescent microscopy for locus visualization, and ultimately co-localization of alternative structures with the PKD1 locus.
Klover, Casey, "Development of a Fluorescent Method for Locus-Specific Targeting of the PKD1 Gene" (2022). Honors Theses. 3617.