Author Date

2020-7

Degree Name

BS

Department

Chemistry and Biochemistry

College

Physical and Mathematical Sciences

Defense Date

2020-07-28

Publication Date

2020-08-04

First Faculty Advisor

Jeffery S. Tessem

First Faculty Reader

Jonathon T. Hill

Honors Coordinator

Merritt B. Andrus

Keywords

INS-1, Beta Cell, CRISPR-Cas9, CRISPR sgRNA Library, Diabetes, Forward genetic screen.

Abstract

By the year 2040, an estimated 642 million people are expected to have diabetes globally. Diabetes results from an elevation of metabolic stressors, such as glucotoxicity, lipotoxicity, oxidative stress and apoptosis. In type 2 diabetes, these stressful conditions contribute to the malfunction and loss of functional insulin-producing β-cells. Current treatment methods for diabetes include insulin therapy, islet transplant and anti-diabetes medication. These treatments are not curative and ignore other factors that contribute to the pathogenesis of diabetes beyond insulin resistance and islet β-cell failure. Previous research on β-cells has focused on ways to replace functional β-cell mass, trigger β-cell proliferation, protect β-cells from stressors and enhance glucose-stimulated insulin secretion. While these treatments are effective, they neglect the possibility of unknown contributing factors. A more effective method is to probe the genetic variation presented in diabetics so that scientists can understand the disease better and develop curative methods. One way of doing this is through gene editing using clustered regular interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9(Cas9).

CRISPR-Cas 9 is a gene-editing tool that modifies DNA using a guide RNA and an endonuclease called Cas9. The guide RNA directs the Cas9 protein to cut a target DNA sequence that is complementary to its RNA sequence. This guided mechanism makes CRISPR-Cas9 the most precise tool for genetic manipulation. Because of its convenience, CRISPR-Cas9 has been used to create many forward genetic screens. Using this knowledge of the connection between β-cells and diabetes, and the CRISPR-Cas9 mechanism, we enzymatically generated a CRISPR guide-RNA library of the rat pancreatic islet β-cell insulinoma (INS-1) cell line. This library can be used to create a CRISPR-Cas9 knockout (KO) forward genetic screen of all the genes in the INS-1 cell line required for insulin secretion, β-cell viability, proliferation, and growth. In the future, this screen will help us identify genes involved in the various mechanistic pathways that contribute to diabetes. For the library synthesis, we will use the total RNA from INS-1 cells to generate the guide RNA libraries.

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