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Title:New tools for live cell imaging of endogenous loci in mammalian cells
Author(s):Tasan, Ipek
Director of Research:Zhao, Huimin
Doctoral Committee Chair(s):Zhao, Huimin
Doctoral Committee Member(s):Chen, Jie; Chen, Lin-Feng; Zhang, Kai
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Genome editing
microscopy
Abstract:Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and transcription activator-like effectors (TALEs) were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. The focus of my studies was to develop an easy, efficient and scalable method for tagging any endogenous locus of interest for live cell imaging. In the first section (Chapter 2), I described a method called SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat), which I developed for tagging and live cell imaging of non-repetitive, endogenous chromosome regions. This method requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized Tet operator (TetO) repeat and its visualization by TetR-EGFP expression. Using an optimized, irregular TetO repeat, PCR-based creation of donors with short homology arms was feasible and simplified the protocol. I also showed the feasibility of knock-in into heterochromatin regions. In Chapter 3, I focused on characterization and live cell imaging applications of the SHACKTeR method. After validating that integration of TetO repeats did not affect the sub-nuclear localization of the tagged locus, I showed that SHACKTeR enables capturing the dynamics of the interactions of euchromatic and heterochromatic loci with their associated nuclear compartments nuclear speckles or lamina, respectively. As another live cell imaging application, I performed visualization of the replication timing of a specific locus and demonstrated the feasibility of long-term imaging using SHACKTeR. During the replication timing studies, I also demonstrated that TetO repeats do not cause cell cycle defects. As a final control experiment, I showed that TetO repeats do not cause H3K9me3 heterochromatin. In the second half of my dissertation, my aim was to expand the applications of SHACKTeR to study genome organization, for which multi-color imaging of various loci simultaneously would be essential. In Chapter 4, I demonstrated a two-step cloning strategy to create an orthogonal irregular TetO mutant repeat array for two-color imaging of endogenous loci. To reduce the assembly time of repeat arrays, an optimized “bridge oligonucleotide-mediated ligation” protocol was used for de novo assembly of 8-mer repeat arrays, which were then combined into the final 96-mer repeat array via Golden Gate reaction. In Chapter 4 I also compared the fluorescent repressor-operator-based imaging to the dCas9-based imaging method. Simultaneous multi-color imaging of endogenous loci in live cells would allow many studies such as examining various enhancer-promoter interactions or labeling two loci on the same chromosome to determine intrachromosomal distances. Another utility of the two-color imaging would be live cell imaging of the two alleles of a gene with two different colors. A method to label each allele with a different color allows real time tracking of the differences between alleles such as transcription or replication timing. To demonstrate the utility of two-color imaging, in Chapter 5 I described my efforts to visualize allele-specific replication timing. In Chapter 6, I described my preliminary results for a new method for accurate gene targeting. My aim was to engineer a novel linear double stranded donor DNA with protein-capped ends in an effort to prevent random integration of the donor DNA.
Issue Date:2020-07-16
Type:Thesis
URI:http://hdl.handle.net/2142/108684
Rights Information:Copyright 2020 Ipek Tasan
Date Available in IDEALS:2020-10-07
Date Deposited:2020-08


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