Binding Site Selection and Cleavage Activity of Zinc Finger Nucleases Targeting Gaucher Mutation

Abstract

Zinc Finger Nucleases (ZFNs) are one of the most promising tools of gene editing as they can create double stranded breaks (DSBs) at specific sites in the genome. Since ZFNs can be used for producing DSBs, they can be used for targeting specific mutations. In this project, the targeted mutation was a G→A point mutation. This change in the GBA gene (G1226) produces a mutant Glucosidase that is unable convert glucosylceramide into glucose and ceramide. Consequently, the substrate accumulates in the macrophages of liver and spleen manifesting into Gaucher Disease. The goal of this project is to design and assess zinc finger nucleases that specifically target the G1226 mutation. First of all, a target sequence that included the zinc finger binding sites (left and right) flanking a 6 bp spacer (that contained the mutation) was designed and integrated into the yeast genome. Zinc fingers were designed in two rounds of PCR and cloned. These fingers were tested for their binding specificity using Bacterial One Hybrid (B1H) Assay; the cleavage activity of the nucleases was determined using Yeast Nuclease Assay (YNA). The results for both of these assays are inconclusive and inconsistent at this moment. If this project is successful, these nucleases can be used in mammalian models for further investigation. The success in mammalian models will then encourage engineering of novel nucleases that can treat diseases like Gaucher Disease.