Date of Award
Doctor of Philosophy (PhD)
Molecular Science and Nanotechnology
Genome engineering relies on DNA modifying enzymes that are able to locate a DNA sequence of interest and initiate a desired genome rearrangement. Currently, the field predominantly utilizes site-specific DNA nucleases that depend on the host DNA repair machinery to complete a genome modification task. We show here that genome engineering approaches that employ self-sufficient, versatile site-specific DNA recombinase Flp and Cre can be developed into promising alternatives. We demonstrate that a Flp variant evolved to recombine an FRT-like sequence FL-IL10A, which is located upstream of the human interleukin-10 gene, can target this sequence in the model setting and native HEK293 cells. Similarly, Cre variant evolved to recombine at loxP-like sequence LL-69058, which is located upstream of the beta globin gene, can target this sequence in the model setting of CHO cells and human HEK293 cells. The target-specific Flp variant is able to perform the integration reaction but the efficiency of the integration reaction in human cells can be enhanced by `humanizing' the Flp variant gene and by adding the NLS sequence to the recombinase. Cre variant displays a poor replacement activity in the mammalian cells and thus is fused with TAL DNA-binding domain to enhance their performance. TAL-Cre variant is able to perform efficient replacement reaction, when paired with another recombinase (dual RMCE). The Cre variant replacement activity is observed only when they are fused to the TAL
Shah, Riddhi, "" (2016). Dissertation. 110.