Date of Award

Winter 2020

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Jeffry L. Shultz

Abstract

Restriction digests are a commonly utilized process for cleaving DNA at specific, but relatively common sites. Restriction enzymes have widespread use in DNA manipulation. CRISPR/Cas9 is a recently identified endonuclease which utilizes a customizable guide sequence to recognize and cut specific ~20 bp sites located in a DNA sequence. This preliminary research aimed to exploit the potential benefit of DNA restriction using the CRISPR/Cas9 procedure through alterations of different components involved in that system. We sought to refine existing CRISPR/Cas9 protocols and make a budget friendly, user-selectable CRISPR/Cas9 restriction digest protocol. The motivation for this research was to simplify and adapt known CRISPR protocols in hopes of using CRISPR as a targeted restriction enzyme. This project yielded negative results, however, important insights into the dilution of target sequences was achieved.

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