DEVELOPMENT OF A CRISPR-CAS9 CONSTRUCT AIMED AT KNOCKING OUT SETD2 IN U2OS CELLS
Open Access
Author:
Schultheis, Nicholas Charles
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
Yanming Wang, Thesis Supervisor Dr. Lorraine C Santy, Thesis Honors Advisor
Keywords:
CRISPR CAS9 SETD2 U2OS H3K36me3
Abstract:
SETD2 is a methyltransferase associated with chromatin remodeling that activates areas of the genome for gene expression. SETD2 is the sole trimethyltransferase capable of trimethylating H3K36, and in doing so allows RRM2 to be transcribed, a subunit of ribonucleotide reductase. SETD2 loss is found in a multitude of cancers and has far-reaching effects within them, often allowing them to proliferate and avoid p53-mediated apoptosis. CRISPR is a recently-discovered gene-editing technology that offers higher fidelity and more flexibility with regards to available targets. The purpose of this experiment was to develop a CRISPR-Cas9 product aimed at knocking out SETD2 in U2OS cells. This purpose appeared to have been met, as shown by markedly decreased H3K36 trimethylation. The CRISPR-Cas9 construct appeared to create a cell line of U2OS cells that were heterozygous for the wild-type SETD2 allele, as while the cell line demonstrated lesser H3K36 trimethylation, not all of it was lost.
