Developments in Human Pluripotent Stem Cell Genome Engineering and in Situ Sequencing Technologies

Abstract

Technology is a key driving force in the advancement of scientific discoveries. While DNA sequencing uncovered the blueprint of life encoded in the human genome, functional roles of sequence variants remain largely unknown. This thesis focuses on developing enabling technologies with broad applications for the study of genetic variations and gene regulation. Recent advances in CRISPR/Cas9-based genome engineering technology have revolutionized biomedical research. The facilitated genome editing system employs a programmable RNA that guides the Cas9 nuclease to its target DNA. Furthermore, gene targeting in human induced pluripotent stem cells (hiPSCs) offers the unprecedented potential for dissecting gene function and correcting disease mutations to fulfill the vision of personalized regenerative medicine. Despite phenomenal progress, the efficiency of targeted modifications remained low in hiPSCs. In part I of this thesis, we developed an efficient genome editing platform by reversibly integrating doxycycline-inducible Cas9 into the genome (iPS-Cas9). We characterized and optimized critical parameters for efficient targeting, generated precise mutations for disease modeling, and demonstrated the potential of multiplexed and continuous editing. Additionally, we initiated efforts to improve homology directed repair (HDR) frequency relative to nonhomologous end joining (NHEJ) via coupling strategies. This versatile platform enables rapid generation of mutant hiPSCs for the study of genome function and provides a test bed for further engineering of Cas9-based tools. While DNA stores the genetic code to life, gene regulation inferred from RNA expression defines cell identity and function. Transcriptome analysis is essential for understanding developmental regulations of complex organisms by deducing gene function from expression pattern and detecting altered gene expression in disease. Traditional gene expression assays are limited by the lack of specificity, spatial context, single-cell resolution, or scalability. In part II, we explored two strategies – padlock probe (PLP) and fluorescence in situ sequencing (FISSEQ) – to develop highly multiplexed in situ RNA sequencing with single cell resolution. We concluded that PLP-based method is suitable for targeted analysis of few transcripts, while FISSEQ represents a transcriptome-wide method for in situ RNA profiling. The technologies presented will greatly accelerate the understanding of gene regulation in complex biological samples with broad applications in biology and medicine.