Type of Thesis
Molecular, Cellular, & Developmental Biology
There are 9 Let7 family members present in keratinocytes, all acting as translational suppressors of the same target mRNAs. This redundancy implies that Let7 is crucial to cellular functioning, and it has indeed been found to act as a tumor suppressor, down regulating functions such as proliferation, cell motility, and de-differentiation. I hypothesized that increasing loss of Let7 loci would lead to progressively more tumor-like phenotypes in the cells. Phenotypic analysis of these cells has not yet been possible, but initial Let7 knockouts were successful.
The amyloid precursor protein is found on chromosome 21 and is strongly upregulated in individuals with Down Syndrome. This protein level is thought to play a role in the early onset development of amyloid-beta plaques in the brains of those with DS, one of the hallmarks of Alzheimer's Disease. APP was therefor chosen as a target for CRISPR, not to knock it out, but to delete one copy and reduce its gene dosage to normal levels. This would allow us to assess protein and RNA levels between trisomic and disomic APP, against a background of trisomy 21. If successful, our work will serve as a proof of principle for single knockout of any chromosome 21 gene. To attempt this, we need transient transfection, which is difficult to achieve with plasmids that can last in the cells for weeks. We hypothesized that brief periods of editing, and therefor incomplete knockouts, could be generated by transfecting cells with CRISPR RNAs.
Janiszewski, Lara, "Novel Genome Editing With CRISPR-Cas9" (2015). Undergraduate Honors Theses. 885.