This work was designed as a proof-of-principle concept or prototype to show the effect of protein-assisted targeting of DNA to specific genomic loci. Two strategies were employed to deliver the DNA with the aim that once inside the cell the DNA would be delivered to the target sequence by the assistance of a protein. In our case, the chosen protein was the site-specific meganuclease I-SceI. The first strategy described herein was to bind the targeting DNA to I-SceI by the use of a fusion protein between I-SceI and a known DNA-binding domain, the GAL4-DBD. The second strategy involved using a DNA aptamer to I-SceI to link the targeting DNA and I-SceI. Testing in vivo revealed that in our human cells (HEK-293) single-stranded DNA was more efficient at gene targeting than double-stranded DNA. In order for the first strategy to work, we needed to have some region of double-stranded DNA. We found that in human cells, it was better for gene targeting to have that double-stranded DNA on the 5’ side of our targeting DNA. We also used gel shift assays to confirm binding by our candidate DNA-binding domain, the GAL4-DBD. We were unable to detect expression of the fusion protein of I-SceI and the GAL4-DBD. For the second strategy we were able to construct an aptamer to I-SceI using a variant of the systematic evolution of ligands by exponential enrichment (SELEX). The I-SceI aptamer was synthesized as part of a longer DNA molecule containing homology to a target locus. Using this chimeric oligonucleotide (part aptamer, part DNA repair region) testing was done in both yeast and human cells. Aside from instances where the aptamer’s secondary structure may have been compromised, the aptamer containing oligonucleotide stimulated repair at a rate 2 to 15-fold higher than the non-selected control sequence. These experimental results show that by delivering targeting DNA within close proximity to the site of modification, gene targeting frequencies can be increased.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/52907 |
| Date | 12 January 2015 |
| Creators | Ruff, Patrick |
| Contributors | Storici, Francesca |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
| Format | application/pdf |
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