CRISPR-based functional genomics to study gene regulatory architecture and consequences of genetic variation

Abstract

If we divide the human genome into its noncoding and coding parts, the noncoding portion takes up ~98-99% of the genome, and the coding portion takes up ~1-2% (ENCODE Project Consortium, 2012). The coding portion of the genome contains the genetic template for RNA transcription and protein translation, and translated proteins are the functional units of the cell that carry out biological function. Despite coding genes acting as templates for RNA transcription and protein translation, the noncoding genome contains the vast majority of sequences that regulate the expression of genes (some regulatory sequences reside in coding regions). The classes of sequences that make up regulatory sequences include promoters, enhancers, silencers, insulators, and repressors, amongst others. Proper regulation of genes at the right time and in the right cell types lies at the core of proper cellular function, and consequently, healthy cells, tissues, and organisms. Variants such as single base substitutions, deletions, and insertions, can occur in both the coding and noncoding portions of the genome. These variants are most often benign. However, some mutations are loss-of-function mutations, meaning that they interfere with and sometimes inhibit proper biological function. Understanding the functional consequences of these mutations is critical to understanding the genetic causes of diseases as well as to develop therapeutics to treat diseases that have a genetic cause. To study both coding and noncoding regions of the genome, novel methods and technologies are required. In particular, the development of methods that can perturb and assess genetic sequences at scale are needed to tackle the vast number of genes and regulatory sequences that the genome consists of, which is on the scale of tens to hundreds of thousands (ENCODE Project Consortium, 2012). In the first chapter, I introduce the field of DNA sequencing, genomics, and genomic technology development. I also discuss the various CRISPR/Cas9-based perturbation methods and how these methods can be utilized to develop novel screening methods, two of which I developed during my PhD. In the second chapter, I describe multiplex, single-cell CRISPR activation (CRISPRa) screening, a method I developed during my PhD and utilized to identify both proximal and distal cell type-specific regulatory elements that are capable of increasing target gene expression when activated via a specific CRISPRa perturbation. In the third chapter, I introduce a CRISPR prime editing based method that allows for the identification of drug resistance mutations in a multiplex and scaled manner. In the fourth and final chapter, I describe how I envision this work advancing further in order to gain a more comprehensive understanding of gene regulatory architecture and the functional consequences of genetic variation in all noncoding and coding sequences. I conclude by discussing how we can use this understanding to design and develop novel and effective therapies for the wide range of diseases that have genetic causes.