Electronic Thesis and Dissertation Repository


Master of Science




Dr David Edgell


Tev-mTALENs are genome-editing nucleases which combine the nuclease and linker domains of I-TevI with the DNA-binding domain of a TAL effector. The linker domain interacts with a portion of the Tev-mTALEN target site called the DNA Spacer, facilitating DNA cleavage. Linker-DNA Spacer interactions are poorly understood but necessary for Tev-mTALEN activity. I examined the DNA Spacer sequence requirements of the linker by assaying Tev-mTALEN activity on targets with mutated DNA Spacer sequences. I also performed activity assays using Tev-mTALENs with mutations to the I-TevI linker domain. My results indicate that the linker DNA Spacer sequence requirements are highly cryptic. No single nucleotide requirements exist at any position in the DNA Spacer. However, assays with mutant Tev-mTALENs have shown that small amino acid mutations to the linker domain can alter or relax the sequence requirements of Tev-mTALENs, increasing their targeting potential.