Genome Engineering Tools to Dissect Gene Regulation

Over the past several years genome and epigenome engineering has been propelled forward by CRISPR-Cas technologies. These prokaryotic defense systems work well in mammalian cells in a manner that is remarkably robust: they are non-toxic, fold into a catalytically active state, localize to targeted cellular compartments, and act on the eukaryotic genome, which is heavily compacted in chromatin. While all these are true, CRISPR-cas nucleases did not evolve to function as highly specific genome engineering tools. Thus, the major goals of the work presented herein are to i) refine the specificity of CRISPR-Cas enzymes, ii) develop methods that facilitate genome engineering in human cells, and iii) apply these technologies toward outstanding problems in human gene regulation. With regard to the first goal, we set out to develop a method that could be easily applied to increase the specificity of diverse CRISPR systems. Adopting RNA-engineering to achieve this goal, we modulate the kinetics of DNA strand invasion to increase the specificity of Cas enzymes. Since the guide RNA is a feature that is common across all CRISPR systems, we expect that this new method to tune the activity and specificity of Cas enzymes will be broadly useful. To address the second goal, we set out to develop an experimental pipeline for the high throughput, precise modification of mammalian genomes. Specifically, we modify the C-termini of genes to include an epitope tag for the genome-wide profiling of transcription factor binding sites. We apply this method to over 30 genes, encoding a variety of transcription factors, chromatin modifying enzymes, and gene regulatory proteins. Out of the large number of genes we focus particularly on members of the AP-1 transcription factor family and nuclear receptor co-activator and co-repressor families. Using this ChIP-seq data, which profiles genome wide binding, and integrating a variety of other genomic information, including chromatin modifications, chromatin accessibility, other TF binding, and inherent regulatory activity, we investigate the dimerization preferences of AP-1 subunits, their genomic binding patterns, and the regulatory potential of theses subunits. Toward addressing the third goal, we decided to focus on the glucocorticoid receptor (GR). The dual activating and repressive function of the GR is incompletely understood, and this duality is a property of many other stimuli responsive transcriptional responses (e.g. NFKB signaling). Thus, how one transcription factor is biochemically endowed with the ability to both activate and repress gene expression is an outstanding problem in gene regulation. It is hypothesized that the GR recruits a variety of distinct protein complexes in order to mediate its diverse function. We used CRISPR based loss of function screening in order to discover new GR cofactors. Using this method, we find a number of cofactors, both canonical and novel, that regulate this response in A549 cells. Ongoing work investigates how general these cofactors are across the transcriptome and whether they provide an avenue to decouple GR’s dual function, which has been a major goal in drug development. Through these studies we have found a way to make CRISPR systems more specific, developed and applied CRISPR based method to define AP-1 binding and function, and used unbiased CRISPR based screens to discover novel regulators of the glucocorticoid drug response.

Chapter 1 broadly introduces this work, its motivations, and aims of research presented herein.

Chapter 2 provides an introduction to both genome engineering and gene regulation. Specifically, it describes the development and application of CRISPR-cas tools and details outstanding problems in gene regulation through the lens of nuclear receptors.

Chapter 3 describes the purification of Cas9 protein and its characterization biochemically. Specifically, we use AFM to determine the DNA binding properties of Cas9 in vitro.

Chapter 4 introduces a new method to modulate the specificity of CRISPR systems in human cells. Therein we show that RNA secondary structure can be applied to diverse CRISPR systems to tune their activity.

Chapter 5 details a method for the high throughput tagging of transcription factors. It specifically investigates members of the AP-1 transcription factor complex.

Chapter 6 is an investigation of the glucocorticoid receptor and its cofactors. We apply a variety of genome engineering and genomic methods to characterize known co-factors and discover new ones.

Chapter 7 is an outlook on the fields of genome-engineering and gene regulation. It describes key questions that are still unanswered and possible lines of attack to address them.