CHARACTERIZATION OF THE TYPE I-E CRISPR SYSTEM OF T. FUSCA AND ITS APPLICATION TO GENOME EDITING
Dolan, Adam Emin
Genome editing techniques and platforms for nucleic acid targeting have been revolutionized by the discovery and technological adaptation of bacterial immune systems called CRISPR systems. These immune systems can specifically recognize, bind to, and cleave substrate nucleic acids to prevent phage infection for the bacterial strains which contain them. In genetic manipulation technologies, CRISPR systems allow for a high degree of targeting versatility along with simplified experimental design compared to other platforms. While Cas9 technologies continue to prove successful, other CRISPR systems exist which deserve investigation for their unique biochemical properties. In this thesis, I first provide background on the mechanisms of CRISPR biochemistry. I focus primarily on the Type I-E CRISPR systems of E. coli and T. fusca (Cascade and Cas3) and make analogies to the well-characterized Type II-A system of S. pyogenes (Cas9). I provide some background on the discovery of CRISPR systems and touch on the organization and diversity of these immune systems in nature. I then also discuss some current trends in CRISPR technology, and since most of these technologies utilize the Cas9 platform, I discuss Cas9 in more detail. Then I explore the mechanism of interaction between Cas3 and Cascade by mutational interface perturbation. The linker-helix region of Cas3 has been established to be important for the interaction between Cascade and Cas3. However, the precise mechanism was difficult to determine without a detailed structure of Cascade interacting with Cas3. While there now exists a Cascade-Cas3 high-resolution Cryo-EM structure, my mutational analysis confirms that the interface at the linker helix is extensive and redundant. However, my analysis also suggests a more nuanced and not completely understood mechanism, since there were several mutations which caused observed binding defects which did not participate in the Cascade-Cas3 interface in the Cryo-EM structure. Next I describe a genome editing technology based on the Type I-E CRISPR system of T. fusca which we applied in a human embryonic stem cell (hESC) line. When applied to hESCs, the Type I-E system introduces a spectrum of deletions that are caused by Cas3 and Cascade. These deletions have non-determinate start and stop sites, ranging from several hundred bases to many kilobases deleted. These deletions did not start at the targeting site, implying that some distance of Cas3 translocation is required before double-strand breaks are induced. Finally, I describe efforts to elucidate the mechanism of Cas3-induced double-strand breaks. The observation that DSBs form distal to the target site in Cascade/Cas3-mediated genome editing implies a new mechanism of Cas3 activity. I present speculation and preliminary data which suggests that Cas3 dimerization is a plausible hypothesis to explain this behavior and I provide a road-map for investigation.
Biochemistry; Molecular biology; Cas3; Cascade; CRISPR; Genome Editing; Bioengineering
Alani, Eric E.; Lis, John T.
Biochemistry, Molecular and Cell Biology
Ph. D., Biochemistry, Molecular and Cell Biology
Doctor of Philosophy
dissertation or thesis