Undergraduate Honors Theses

Thesis Defended

Spring 2018

Document Type

Thesis

Type of Thesis

Departmental Honors

Department

Biochemistry

First Advisor

Dr. Deborah Wuttke

Second Advisor

Dr. Joel Kaar

Third Advisor

Dr. Ryan Gill

Fourth Advisor

Dr. Jeff Cameron

Fifth Advisor

Dr. Xuedong Liu

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Abstract

CRISPR/Cas9, originating from a bacterial immune system, has been identified as a powerful tool for genome modification (Jinek, et al., 2012). Historically, analyzing genotype-phenotype relationships have been limited by DNA synthesis and sequencing technology. Recent developments in genome editing (CRISPR/Cas9) coupled to improvements in synthesis and sequencing allows mapping these relationships at an entirely new scale. A technique novel to the Gill lab for genome modification that harnesses the CRISPR-Cas system is “CRISPR Enabled Trackable Genomic Engineering” or “CREATE” technology. CREATE technology is one such strategy for simultaneously making hundreds of thousands of single genomic edits, then tracking the resultant phenotypes. In this study, I alter parameters of the CREATE mechanism to elucidate the cause of variability in editing efficiency between different editing constructs. I demonstrate that editing efficiency is impacted by guide RNA toxicity, genomic position, expression levels of Cas9, availability of template for homologous recombination, and expression levels of phage-based recombination proteins. I find that high toxicity is related to high editing efficiency. The position of the gene being edited in the bacterial genome impacts editing efficiency in a pattern dependent on guide toxicity. Expression levels of Cas9 also affect editing efficiency in a guide toxicity-dependent fashion. Additional results studying recombination suggest increasing intracellular abundance of donor DNA can lead to increased editing efficiency, in a genomic position-specific manner. A final finding was that expressing recombination proteins using a constitutive promoter replicated the behavior of the traditional lambda Red expression system, opening up possibilities for future modifications.

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Biochemistry Commons

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