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Join us as PhD in Applied Life Sciences student Michael De La Cruz speaks about targeted genome-scale gene activation and gene editing in human cells to understand disease models.
“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. Functional genomic screens with CRISPR-Cas9 based genomic tools facilitate the high-throughput precise manipulation of genes, allowing unbiased analysis of phenotypic consequences. We used 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 eleven potential candidate genes that can be targeted to prevent resistance to a 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 the pathogenesis of HD, 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 expressing both a red fluorescent protein (RFP+) and a mutant green fluorescent protein (GFP–, which can become GFP+ by the nucleolytic activity of the TALENs), and donor DNA with part of the mutant (high-CAG) HTT gene. The reporter plasmid allowed for transient selection of RFP+ transfected clones and confirmation of nucleolytic activity in GFP+ clones. Single RFP+ and GFP+ cells were FACS-sorted into individual wells and subcultured. Capillary gel electrophoresis indicated the presence of high CAG allele at an estimated targeting frequency of (without the use of any selectable marker) of 29%.”
Monday, January 28, 2019 at 10:30 a.m.
Building 535, East Conference Room