Date Degree Awarded
Open Access Dissertation
PHD in Applied Life Sciences
First Thesis/Dissertation Advisor
Prof. Animesh Ray
Second Thesis/Dissertation Advisor
Prof. Ian Phillips
Since the discovery of sequence directed DNA editing reagents such as CRISPR-Cas9 RNA-guided and TALEN DNA endonucleases, there has been a snowball of advances in the life sciences due to the ability to efficiently edit and control genomes within living cells. CRISPR-Cas9 based genomic tools, which facilitate the high-throughput precise manipulation of genes, allow for unbiased functional genomic screens. We used a human CRISPR-Cas9 Synergistic Activation Mediator pooled library which utilizes an engineered protein complex for transcriptional activation of 23,430 endogenous genes to investigate the development of novel resistance mechanisms to lung cancer targeted therapy, Erlotinib. We set out to identify genes that when activated cause resistance to Erlotinib, with the ultimate aim to develop parallel therapies to systematically inhibit the pathways that these genes control or their product so as to prevent the evolution of drug resistance. Unlike current methods, these genes, when targeted, should not affect cancer cell metabolism, thereby decreasing the chances for cytotoxic effects. We have identified at least six potential candidate genes that could be targeted to prevent resistance to tyrosine kinase inhibitor, Erlotinib. In a separate study, we attempted to develop an isogenic (same genetic background besides the disease mutation) Huntington’s Disease (HD) human cell lines through TALEN mediated gene editing. Multiple cellular pathways have been implicated in HD pathogenesis, but normal function of the gene, essential for embryogenesis in mouse, has remained controversial. Moreover, the effects of genetic variation at other loci on the abnormal Huntingtin protein toxicity have been indicated, yet remain poorly studied. An isogenic set of HD cell lines should allow for an unbiased look into these effects. HEK293 cells were co-transfected with TALEN expression constructs, a reporter plasmid, and donor DNA with part of the mutant (high-CAG) HTT gene. The reporter plasmid allowed for selection of transfected clones (RFP+) and confirmation of nucleolytic activity in clones (eGFP+). RFP+ and eGFP+ cells were FACS-sorted into individual wells and subcultured. Trinucleotide Repeat Sizing analysis indicated the presence of high CAG allele at an estimated targeting frequency (without the use of any selectable marker) of 38% for the FACS selected cell lines.
© 2019 Michael De La Cruz
De La Cruz, Michael. (2019). Targeted Genome-Scale Gene Activation and Gene Editing in Human Cells to Understand Disease Models. KGI Theses and Dissertations, 23. https://scholarship.claremont.edu/kgi__theses/23.