Feng Li

Date of Award

Spring 5-2020

Document Type


Degree Name

Doctor of Philosophy (PhD)


Molecular Science and Nanotechnology

First Advisor

Yuri Voziyanov


In this dissertation, DNA recombinase Flp modified variants were developed for gene editing. The Flp recombinases can perform recombination reactions to modify the DNA sequence on the Flp recombination target (FRT). The Flp can recognize the DNA sequences,which resemble the wild-type FRT when modified. The Flp modified variants show potential values for genomic engineering because such FRT-like sequences as targets are widespread in the human genome. Our goal is to evolve the Flp variants on FRT-like targets in the human genome for safety and efficiency, correcting the genome mutations which cause the genetic disease.

Gene editing relies on site specificity nucleases such as transcription activator-like effectors nuclease (TALEn), Zinc finger nuclease, or CRISPR/Cas9. Flp, as a site specificity recombinase has different mechanisms on the DNA modification. The recombinases do not cut the DNA by removing the fragment as nucleases and show better safety in gene modification. That's the reason we chose site specific recombinases as our research target.

We chose the FRT-like sequences in the human interleukin gene and beta hemoglobin gene as our target for Flp evolving. To improve the specific activity of these Flp variants on targeted FRT-like sequences in human cells, DNA-binding domain from transcription activator-like effectors (TALE) which are found in the plant pathogen Xanthomonas were fused to the Flp variants, to form Flp-TAL variants. The DNA-binding domain from TALE can be programmed to recognize the designated DNA sequence and improve the specificity binding of the nearby working domain.

The Flp-TAL variants have been proven able to perform DNA recombination in human cells. My work concerns evolving Flp-TAL variants to recombine the FRT like sequences that are located in the human gene, and testing them under model conditions in human cells.