Date of Award

1-2019

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Carlos A. Molina

Committee Member

Robert Meredith

Committee Member

Mitchell Sitnick

Abstract

Inducible cAMP Early Repressor (ICER) is a small transcription factor that originates from an intronic promoter within the cAMP Response Element Modulator (CREM) gene (Molina et al., 1993). ICER acts as a putative tumor suppressor by mediating cAMP antiproliferative activity by competitively binding to cAMP Response Elements (CRE’s) and repressing transcription of genes involved in cell division (Mémin et al., 2002). ICER has been shown to be effectively absent in cancer and suspected to be targeted for proteasomal degradation via ubiquitination (Healey et al., 2013). ICER is most likely regulated via post-translational modifications as a result of mutations on Ras/Raf oncogenes (Healey et al., 2013). For example, it has been shown that in cancer, mutant activated GTP bound Ras protein continuously activates Braf, which in turn, results in over activation of the Mitogen Activated Protein Kinases, ERK1/ERK2 (Zhang and Liu, 2002). ICER is thought to be phosphorylated by ERK1/ERK2 and subject to proteasomal degradation; a result of ubiquitination in Ras/MAPK-mediated melanoma tumorigenesis (Healey et al., 2013). Melanoma cells rapidly apoptosed when transfected with a mutant form of ICER that did not contain any lysine residues. This was most likely because the transcriptional repressor ICER was unable to be ubiquitinated. While this highlights ICER’s importance in cell division and growth, it is challenging to study the effects of ICER in melanoma if cells are unable to survive. One method to circumvent this issue is to create an inducible cell line, in which a mutant form of ICER with no lysine residues is under a promoter whose expression can be toggled on and off, dependent on the presence of a transactivator. This requires that mutated ICER be knocked-in to an alternative location in the human genome, and that wildtype ICER be efficiently knocked-out in order to solely study the effects of the mutant. In this experiment, CRISPR Cas9 mediated genetic editing was used to knockout ICER, while mitigating off target effects on CREM gene, in an attempt to maintain otherwise normal cell physiology. The target loci of interest in this experiment was the Kozak consensus on an ICER specific promoter and the target of the guide RNA (gRNA) for Cas9 endonuclease activity. Amplicon Libraries of DNA extracted from cells transiently transfected with plasmid containing gRNA and Cas9-GFP cassette and empty vector expressing only EGFP (control) were generated with Nextera index adaptors. Paired-end sequencing on the Illumina Miseq provided sufficient coverage depth to determine how efficient the gRNA was at generating insertions/deletions (indels) or substitutions at the desired loci. Sequencing data between Experimental and Control was first reviewed using CRISPResso, an online bioinformatic tool that analyzes deep sequencing data. An additional bioinformatic analysis was designed and performed to corroborate CRISPResso results and identify any other low-level variants.

The goal of this experiment was to develop the workflow to identify possible indels/substitutions that resulted from CRISPR Cas9 induced genetic alteration. This was done with the expectation of identifying a possible knockout of ICER, while minimally affecting CREM. Although no variants identified suggest an ICER knockout, a scalable workflow is now in place to facilitate this stage of the experiment.

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