Targeted Gene Editing Approaches to Study Target Genes

Author

• Seth Brian Eddington, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2026

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Davie, Judith

Second Advisor

Burgess, Rebecca

Abstract

The employment of CRISPR systems for generation of gene knockouts, frameshift mutations, and Homology Directed Repair (HDR) insertions in a gene specific manner is not without faults. Off-target effects, editing in locations that are undesired, hinder the safety and effectiveness of the system for clinical gene therapeutic applications. Here we have designed Cis-SNuBRs (Cis-acting small nucleic acid-based regulators) of SpCas9 that inhibit the enzyme and are engineered to be derepressed by an oligonucleotide complementary to any sequence of choice, effectively enabling targeted activation of cleavage activity. Bmi1, a polycomb repressive complex protein, prevents development of ataxia in mice. Bmi1 deficient mice display a decline in motor function and cerebellar architecture defects, but the cell type responsible is unknown. Using tissue-specific Cre models and the Bmi1 floxed conditional allele, we generated a number of mouse lines to investigate the effect of Bmi1 deficiency in different tissues on the development of progressive ataxia. We found that mouse lines with cerebellar deficiency of Bmi1 exhibited neuromotor behavior defects that coincided with thinning of the molecular layer and altered morphology of Purkinje cells and dendrites. We show that Bmi1 prevents Purkinje cell layer degeneration and mouse motor deficits in a manner independent from the Bmi1 canonical gene targets, p16Ink4a and p19ARF. Finally, we propose application of Cis-SNuBRs to effectively knockout Bmi1 in Purkinje cells in neonatal cerebellar cell cultures in vitro to analyze the specific role of Bmi1 in Purkinje cell morphology and dendrite growth and maturation.