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- All Subjects: Biochemistry
- Creators: Mills, Jeremy
- Status: Published
This thesis focused on the expression and crystallization the fragment antigen binding antibody fragment A4. A fragment antigen binding fragment was chosen to be worked with as it is more stable than many other antibody fragments. A4 is important in Alzheimer’s disease as it is able to identify toxic beta amyloid.
I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.
SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.
Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.
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.