When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.
Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.
Gene editing technologies have facilitated the genetic manipulation of cells to study proteins by generating knockouts, point mutations and endogenous tags. Amongst precise edits, endogenous tags provide valuable tools to study protein localization in live cells. However, precise gene editing is inefficient in mammalian cells, and generating edited cell lines is time-consuming. Therefore, most studies of cellular processes in human cells rely on other methods such as over-expression, and use cancerous or transformed cell lines that are easy to manipulate. For example, cytokinesis, the physical separation of a cell into two daughter cells at the end of mitosis, is mainly understood in the context of cancerous HeLa cells. In this thesis, I present tools and protocols to tag proteins endogenously in multiple human cell lines, which enables the study of cytokinesis proteins in their native cellular context. I first generated endogenous tags using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR-associated protein 9) to study protein localization during cytokinesis in different human cell lines. By characterizing cytokinesis parameters, I found that cytokinesis occurs differently in different cell lines, suggesting that the underlying mechanisms regulating cytokinesis differ with cell type. I then engineered an iPS cell line that facilitates large-scale endogenous tagging by taking advantage of a split mNeonGreen protein where the tagging fragment is short. Endogenous tagging with this system is efficient and can be scaled up for high-throughput editing and screening. This work provides new tools to study protein localization during cytokinesis and other cellular processes in human cells. It also enables comparative studies of cellular processes across human cell types to understand cellular function on the scale of all human cell types.