Submission Title

Use of CRISPR-Cas9 Gene Editing System to Characterize the Human CXCL10 Viral Response Gene

Location

Jereld R. Nicholson Library: Grand Avenue

Subject Area

Biochemistry

Description

Given its ease of use and accurate functionality, the CRISPR-Cas9 gene editing system has been at the forefront of recent genetic and biochemical research ranging from the development of powerful gene therapies to the characterization of novel genes. Here we attempt to characterize the human CXCL10 gene, a secreted chemokine ligand associated with cellular response to viruses, by directed gene mutation accomplished by CRISPR-Cas9. Use of this system requires the design and synthesis of a complete plasmid, containing DNA that codes for all necessary components of CRISPR-Cas9, and amplification of this plasmid in E. coli. Following characterization tests, the amplified plasmid could be transfected into an immortal human lung cell line sample and possibly effect a targeted knockdown of the CXCL10 gene. In this process, we observed evidence of correct CRISPR-Cas9 plasmid construction by PCR amplification followed by gel electrophoresis analysis. We also observed successful transfection of E. coli with our plasmid by ampicillin screening followed by sequencing analysis of the amplified product. Current efforts lie in optimizing lung cell transfection, though recent observations of our puromycin screen indicate some level of desired transfection. After establishing a transfected cell population, our future work will include identification of successful gene knockdown by sequencing CXCL10 where we expect to see a mutation made by CRISPR-Cas9, in addition to performing Western blots to detect possible changes in the CXCL10 protein product. The creation of a CXCL10 knockdown cell line will aid in future tests regarding the functionality of this gene in immune response, as well as demonstrate the exciting potential of CRISPR-Cas9 as both an accessible learning tool and a powerful instrument for new discovery.

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Use of CRISPR-Cas9 Gene Editing System to Characterize the Human CXCL10 Viral Response Gene

Jereld R. Nicholson Library: Grand Avenue

Given its ease of use and accurate functionality, the CRISPR-Cas9 gene editing system has been at the forefront of recent genetic and biochemical research ranging from the development of powerful gene therapies to the characterization of novel genes. Here we attempt to characterize the human CXCL10 gene, a secreted chemokine ligand associated with cellular response to viruses, by directed gene mutation accomplished by CRISPR-Cas9. Use of this system requires the design and synthesis of a complete plasmid, containing DNA that codes for all necessary components of CRISPR-Cas9, and amplification of this plasmid in E. coli. Following characterization tests, the amplified plasmid could be transfected into an immortal human lung cell line sample and possibly effect a targeted knockdown of the CXCL10 gene. In this process, we observed evidence of correct CRISPR-Cas9 plasmid construction by PCR amplification followed by gel electrophoresis analysis. We also observed successful transfection of E. coli with our plasmid by ampicillin screening followed by sequencing analysis of the amplified product. Current efforts lie in optimizing lung cell transfection, though recent observations of our puromycin screen indicate some level of desired transfection. After establishing a transfected cell population, our future work will include identification of successful gene knockdown by sequencing CXCL10 where we expect to see a mutation made by CRISPR-Cas9, in addition to performing Western blots to detect possible changes in the CXCL10 protein product. The creation of a CXCL10 knockdown cell line will aid in future tests regarding the functionality of this gene in immune response, as well as demonstrate the exciting potential of CRISPR-Cas9 as both an accessible learning tool and a powerful instrument for new discovery.