Multiplexed reading and writing of the transcriptome

Abstract

Having complete spatial and temporal information about biological systems will provide the ultimate guidebook about how complex systems like the human body function. In the meantime, the understanding of how the complex human body work will inevitably lead to strategies to perturb, restore, or even optimize how it performs. While the human genome is the underlying blueprint that instructs how the system should work, the transcriptome is the functional manifestation that carries the genetically encoded information into action. Reading the transcriptome could offer valuable insight into cellular function, tissue/organ development, and disease progression. Technologies for reading the transcriptome have evolved tremendously over the past two decades, from microarray to next-generation sequencing (NGS), to single-cell sequencing, and, recently, to spatial transcriptomics. As the methods for reading the transcriptome evolve, so does the methods for writing the transcriptome. From siRNA to transcription factors, from zinc-finger proteins to CRISPR/Cas9, the arsenal to precisely influence and control cellular behaviors has expanded rapidly. In this thesis, work on both fronts will be presented. Chapter One reviews previous developments in technologies for reading and writing of the transcriptome. Chapter Two describes the first major project about developing a highly multiplexed method for targeted in situ RNA detection, Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses (BOLORAMIS). Chapter Three describes the second major project about iterative single-cell transcription factor library screens for hiPSCs differentiation, with a particular focus on microglia. Also included in this chapter is an exploratory analysis of using single-cell atlas data to guide cell fate engineering. Finally, in Chapter Four, conclusions from the work presented in previous chapters and future directions are discussed.