Date Presented

Spring 5-2021

Document Type

Thesis

Degree Name

Bachelor of Science

Department

Biomedical Science

First Advisor

Dr. Erin Friedman

Second Advisor

Dr. Jamie Brooks

Third Advisor

Dr. Domenica Favero

Abstract

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and the Cas9 nuclease enzyme work as an editing software for DNA. Bacteria use this technology to cleave a piece of any foreign DNA entering a cell and render it useless. However, the CRISPR-Cas9 system can be modified to downregulate any targeted gene in an organism. Scientists have already been successful in transforming CRISPR-Cas9 into eukaryotic cells. The system can be programmed with guide RNA to find a specific section of a genome to cleave. This allows for specific downregulation of any target genes or genes in a cell. The model plant Arabidopsis thaliana is an ideal genetic candidate for the use of CRISPR-Cas9 because the full genome has been sequenced, it has a small genome, and it has a short life cycle. Using a floral dipping method, CRISPR-Cas9 will be introduced to Arabidopsis thaliana to downregulate candidate genes that should affect the plant’s phenotype. The main goal of this thesis is to create a system for CRISPR- Cas9 research at the University of Lynchburg that is accurate, precise, and easy to use. This will be done using two genes, TOO MANY MOUTHS (TMM) and VARIEGATED 1 (VAR1), which are two genes with known function and clear phenotypes. Plants in which TMM is mutated display increased stomatal density, and VAR1 mutants contain downregulated genes involved in photosystem II, leading to stunted growth. Once CRISPR-Cas9 can be used successfully at the University of Lynchburg, future researchers can use it to target genes of interest to determine their functions.

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