| The human genome encodes information that instructs human development, physiology, medicine, and evolution. Massive amount of genomic data has generated an ever-growing pool of hypothesis. Genome editing, broadly defined as targeted changes to the genome, posits to deliver the promise of genomic revolution to transform basic science and personalized medicine. This thesis aims to contribute to this scientific endeavor with a particular focus on the development of effective human genome engineering tools.;Chapter 1 introduces the key topics on genome editing, with an emphasis on its implications, current status, and potential applications.;Chapter 2 describes the generation of reTALEs, a simplified form of TALENs, and the assembly of a pipeline to scarlessly edit human stem cells. We demonstrate the utility of this pipeline by generating hiPSCs with mutations in HIV resistance genes within 3 weeks.;Chapter 3 describes the generation of a novel RNA-guided human genome editing tool. We reprogrammed a type II bacterial CRISPR system to function in a human context, and demonstrated an efficient & multiplexable version of this approach in multiple cell types including human iPSCs. We compared the efficiency and specificity of CRISPR with TALE and designed a new strategy to mitigate the off-target issues associated with CRISPR.;To expand our genome editing toolbox, Chapter 4 describes the assembly of novel chimeric deaminases that perform sequence-specific genome editing without generating DSBs and the need to simultaneously provide replacement (i.e., donor) DNA. Targeted deaminases are both efficient and specific in Escherichia coli and human cells, presenting an alternative platform that can eventually be used in multiplex genome editing.;Chapter 5 describes our effort in combining genetically engineered iPSCs with organ-on-chip models to investigate the cellular etiology of disease and to identify potential therapeutic targets. We generated isogenic iPSCs carrying a mutation identified in cardiomyopathy patients. Cardiomyocytes derived from engineered hiPSC recapitulated disease abnormalities and engineered "heart on chip" tissues contracted poorly. Replacement of the defective gene product corrected these abnormalities.;We finally conclude with remarks on the future prospects for genome editing to expand our understanding of fundamental biology and to enhance the wellness of human beings. |
