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- Creators: Barrett, The Honors College
- Creators: Brafman, David
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
This thesis covers two topics. First, I attempt to generate stochastic resonance (SR) in a biological system. Synthetic bistable systems were chosen because the inducer range in which they exhibit bistability can satisfy one of the three requirements of SR: a weak periodic force is unable to make the transition between states happen. I synthesized several different bistable systems, including toggle switches and self-activators, to select systems matching another requirement: the system has a clear threshold between the two energy states. Their bistability was verified and characterized. At the same time, I attempted to figure out the third requirement for SR – an effective noise serving as the stochastic force – through one of the most widespread toggles, the mutual inhibition toggle, in both yeast and E. coli. A mathematic model for SR was written and adjusted.
Secondly, I began work on designing a new genetic system capable of responding to pulsed magnetic fields. The operators responding to pulsed magnetic stimuli in the rpoH promoter were extracted and reorganized. Different versions of the rpoH promoter were generated and tested, and their varying responsiveness to magnetic fields was recorded. In order to improve efficiency and produce better operators, a directed evolution method was applied with the help of a CRISPR-dCas9 nicking system. The best performing promoters thus far show a five-fold difference in gene expression between trials with and without the magnetic field.
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.
A mutation rate refers to the frequency at which DNA mutations occur in an organism over time. In organisms, mutations are the ultimate source of genetic variation on which selection may act. However, a large number of mutations over time can be detrimental to the cell. Mutation rates are the frequency at which these new mutations arise over time. This can give great insight into DNA repair mechanisms abilities as well as the mutagenic abilities of selected factors. CRISPR-Cas9 is a powerful tool for genome editing, but its off-target effects are not yet fully understood and studied. With its increasing implementation in science and medicine, it is crucial to understand the mutagenic potential of the tool. S. cerevisiae is a model organism for studying genetics due to its fast growth rate and eukaryotic nature. By integrating CRISPR-Cas9 systems into S. cerevisiae, the mutational burden of the technology can be measured and quantified using fluctuation assays. In this experiment, a fluctuation assay using canavanine selective plates was conducted to determine the mutational burden of CRISPR-Cas9 in S. cerevisiae. Multiple trials revealed that various strains of CRISPR-Cas9 had a mutation rate up to 3-fold higher than that of wild-type S. cerevisiae. This information is essential in improving the precision and safety of CRISPR-Cas9 editing in various applications, including gene therapy and biotechnology.
The purpose of this experiment is to deliver DNA origami barrels loaded with Cas13d-gRNA binary complexes to HPV-16 and HPV-18 cervical cancer lines to make the cancer mortal. The production of Cas 13d has proven successful with a concentration of ~ 1mg/mL, but the activity assay performed has not shown conclusive evidence of Cas13d and guide RNA binary complex formation or activity. Successful annealing of the DNA origami barrel has been quantified by an agarose gel, but further quantification by TEM is in progress. Overall, steady progress is being made towards the goal of targeting HPV16 E6/E7 pre-mRNA with CRISPR/Cas13d.