Development and application of a general method for somatic cell genetics

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017."June 2017." Cataloged from PDF version of thesis.Includes bibliographical references.Genetic analysis, or genetic screening, is an efficient method for identifying the set of genes involved in a biological process. In microorganisms, powerful techniques enable systematic loss-of-function screens, which have yielded critical insights into the molecular basis of many fundamental cellular pathways. In human cells, however, prevailing screening methods fall short - leaving our understanding of human genes incomplete. Here, I describe the development of a general method for genetic screening in mammalian cells using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. In this approach, libraries of knockout mutants can be generated in any cultured cell line using large-scale lentiviral single-guide RNA (sgRNA) pools and screened under both positive and negative selection. After the initial, proof-of-principle experiments, I constructed a genome-wide sgRNA library and screened for genes required for the proliferation and survival of the nearhaploid chronic myeloid leukemia cell line, KBM7. The unusual karyotype of these cells also allowed an independent screening approach, involving gene-trap insertional mutagenesis, to find cell-essential genes. Together, these screens converged on a highly overlapping gene set that was enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. To broadly survey patterns of human gene essentiality, I conducted CRISPR-based screens across many more cell lines and cancer types. Overall, these studies revealed a large set of common essential genes involved in housekeeping processes but, interestingly, also pinpointed differences specific to each cell line that reflected its developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure. By mapping sets of genes displaying variable but correlated essentiality across lines, I uncovered functionally interacting gene networks. Using this dataset, I also identified genetic vulnerabilities associated with oncogenic Ras which may be exploited for anti-cancer therapy. Last, in collaboration with Bruce Walker and Nir Hacohen's labs, I have conducted a screen for host factors (i.e. human genes) that are required to support HIV infection in a physiologically relevant cell line model. Collectively, these results establish CRISPR-based screens as a powerful tool for systematic genetic analysis in mammalian cells.by Tim Wang.Ph. D