Using CRISPR-Cas9 as a Restriction Enzyme

Author

Zack Crawley, Louisiana Tech University

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.

Recommended Citation

Crawley, Zack, "" (2020). Thesis. 32.
https://digitalcommons.latech.edu/theses/32