CRISPR-Cas Tools for Engineering Genome Structure and Gene Expression

Abstract

This thesis focuses on the development of two distinct sets of CRISPR-Cas tools: one for gene tethering and the other for logic-based transcriptional regulation. The first tool is a CRISPR-based gene tethering tool. The eukaryotic genome has a complex three-dimensional organization that is thought to play an important role in the regulation of eukaryotic genes. However, studies focused on direct causal effects of repositioning a gene have found changes in transcriptional activity in some, but not all, genomic contexts. A limiting factor for many of these studies is the use of hard-coded DNA binding domains in their gene repositioning tools, that require integration of a DNA binding site into every locus of interest. Integration of DNA limits the number of sites that can be tested and potentially changes the genomic context of the locus being tested. Use of CRISPR-Cas systems would enable the DNA integration requirement to be circumvented. To address this, I developed a CRISPR-based tool for tethering genes to the nuclear periphery in yeast. I benchmarked this tool against a previously developed Gal4-based gene tethering tool and demonstrated that both localize genes to the nuclear periphery. Notably, the Gal4-based gene tethering tool also demonstrated a silencing phenotype while the CRISPR-based tool did not. This suggests that small changes in the structure of gene positioning tools can have an impact on the transcriptional effects observed. The second set of CRISPR-based tools I developed are for use in logic-based transcriptional regulation. Boolean-style transcriptional logic systems are appealing for bioengineering purposes because of their modularity and composability. A current limitation is a lack of tools that can operate in parallel and an inability to access complex, higher-order Boolean logic functions. To address this, I have developed a series of single-layer, orthogonal transcriptional logic gates. I demonstrate these gates operate alone and that the NOR and OR gates can operate in parallel. In the future, the ability to combine different single-layer logic systems will enable the construction of complex, higher order logic functions.