Development Of Novel Crispr Technologies For The Precise Genome Editing Of Human Pluripotent Stem Cells PDF Download

Precision genome surgery has the potential to revolutionize the field of personalized medicine through applications ranging from basic science, to creation of in vitro disease models, all the way to the correction of genetic disease in vivo. Although the field has rapidly advanced since the development of CRISPR-based technology, the ability to generate precise edits at multiple locations within the genome remains an outstanding challenge. As a result, edited cell populations as well as isolated clones must be rigorously screened for the presence of undesired mutations before any form of clinical application such as ex vivo cell therapy or in vivo somatic cell editing can be undertaken. Thus, there is a need to increase rates of precise editing and decrease rates of imprecise mutation to improve the clinical viability of gene editing. This thesis aims to develop new technologies to address challenges in precision genome editing and subsequently demonstrate their utility in a human pluripotent stem cell disease model. First, I describe a method to track hundreds of cell populations in real time using high-content analysis, allowing the facile selection of those that have undergone genome editing. Second, I demonstrate a new technology that is capable of tethering together CRISPR ribonucleoproteins and donor DNA to increase the frequency of precise gene surgery. Finally, I combine these technologies to correct mutations in an induced pluripotent stem cell model of Pompe disease, an autosomal recessive disorder. These methods and their applications demonstrate advances in the field of precision genome surgery and represent an important step toward the clinical use of genetic therapies.