Barrett, The Honors College Thesis/Creative Project Collection
Barrett, The Honors College at Arizona State University proudly showcases the work of undergraduate honors students by sharing this collection exclusively with the ASU community.
Barrett accepts high performing, academically engaged undergraduate students and works with them in collaboration with all of the other academic units at Arizona State University. All Barrett students complete a thesis or creative project which is an opportunity to explore an intellectual interest and produce an original piece of scholarly research. The thesis or creative project is supervised and defended in front of a faculty committee. Students are able to engage with professors who are nationally recognized in their fields and committed to working with honors students. Completing a Barrett thesis or creative project is an opportunity for undergraduate honors students to contribute to the ASU academic community in a meaningful way.
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- All Subjects: Biochemistry
CRISPR-Cas based DNA precision genome editing tools such as DNA Adenine Base Editors (ABEs) could remedy the majority of human genetic diseases caused by point mutations (aka Single Nucleotide Polymorphisms, SNPs). ABEs were designed by fusing CRISPR-Cas9 and DNA deaminating enzymes. Since there is no natural enzyme able to deaminate adenosine in DNA, the deaminase domain of ABE was evolved from an Escherichia coli tRNA deaminase, EcTadA. Initial rounds of directed evolution resulted in ABE7.10 enzyme (which contains two deaminases EcTadA and TadA7.10 fused to Cas9) which was further evolved to ABE8e containing a single TadA8e and Cas9. The original EcTadA as well as the evolved TadA8e where shown to form homodimers in solution. Although it was shown that tRNA binding pocket in EcTadA is composed by both monomers, the significance of TadA dimerization in either tRNA or DNA deamination has not been demonstrated. Here we explore the role of TadA dimerization on the DNA adenosine deamination activity of ABE8e. We hypothesize that the dimerization of TadA8e is more important for the DNA deamination than for the tRNA deamination. To explore this, I conducted a urea titration on ABE8e to disrupt TadA8e dimerization and performed single turnover kinetics assays to assess DNA deamination rate of ABE8e’s. Results showed that DNA deamination rate and efficiency of ABE8e was already impaired at 4M urea and completely lost at 7M. Unfortunately, CD measurements at the equivalent urea concentrations indicate that the loss of activity is due to the unfolding of ABE8e rather than the disruption of TadA8e’s dimerization.