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Title:Advancements in genome engineering and applications in mammalian systems
Author(s):Xiong, Xiong
Director of Research:Zhao, Huimin
Doctoral Committee Chair(s):Zhao, Huimin
Doctoral Committee Member(s):Belmont, Andrew S; Harley, Brendan A; Kong, Hyunjoon
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Genome editing
Synthetic biology
Live cell imaging
Gene regulation
Gene therapy
Abstract:In the past three decades, we have witnessed the outbreak of technology development and applications in the field of genome engineering. From zinc finger proteins (ZFPs), to transcription activator-like effectors (TALEs), to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), these programmable DNA targeting proteins have been successfully adapted for genome editing, loci imaging and gene regulations. In Chapter 2, I demonstrated an efficient epitope tagging approach that enabled subsequent chromatin immunoprecipitation followed by sequencing (ChIP-Seq) experiments. Specifically, I combined CRISPR-Cas9 with MMEJ (Micro-homology Mediated End Joining) to genetically engineer an endogenous transcription factor (TF) with a 3xFLAG tag in a human colorectal cancer cell line (HCT116). I genotyped modified clones to confirm the precise tag insertion. The TF binding motifs, typical peak locations and peak calling enrichment were validated from ChIP-seq results. Similar analyses and results were observed among single clones and pooled clones, suggesting colony isolation could be skipped for cell line generation. The highly scalable procedure makes this strategy ideal for ChIP-seq analysis of TFs in diverse species and cell types. In Chapter 3, I emphasized on comparing Tet Operator (TetO)/Tet Repressor (TetR) based imaging method with TALE and CRISPR-Cas9 mediated loci labeling methods. Signal to nuclear noise ratios (SNRs) were analyzed first. The high SNRs indicated all methods should be suitable for live cell imaging applications. Labeled loci positioning to nuclear speckles were found similar among these methods. Orthogonal imaging of paired loci was also confirmed by utilizing two of these methods to target genomic regions separated by 7kb. When targeting the sub-telomeric region, loci labeled by TetR showed slightly higher frequency in the form of doublets than the other methods. When targeting a typical euchromatic region, loci labeled by CRISPR-Cas9 based approach maintained much lower doublet frequency than TetR labeling. Long term live imaging was conducted to track the labeled loci by CRISPR-Cas9 during S-phase, which had approximately an hour delay in replication timing. Moreover, fewer doublets were observed from CRISPR-Cas9 labeling over the live imaging acquisition, implying a possibility that CRISPR-Cas9 perturbed sister locus separation. Generally, these imaging methodologies can efficiently label endogenous sites with high SNR and positioning specificity. CRISPR-Cas9 based method may not be the ideal for cell cycle relevant research due to its potential intervention of replication progression. In Chapters 4 and 5, I switched gear to aim at expanding gene regulation toolbox. In Chapter 4, I coupled three nuclease-deficient Cas9 orthologs from Streptococcus pyogenes (dCas9Sp), Neisseria meningitidis (dCas9Nm) and Staphylococcus aureus (dCas9Sa) with chemically inducible dimerizers GAI-GID, FKBP-FRB and ABI-PYL1 respectively. The dCas9 protein was fused with one of the dimerizers and the other dimerizer was fused with activator domain p65-HSF1. Addition of inducers enabled dimerization which resulted in the recruitment of p65-HSF1 molecules to targeted promoter regions for gene upregulation. These three platforms were tested with high induction efficiency and were able to regulate three model genes orthogonally. Spatial and temporal regulation was also executed to show the applicability of multi-pathway regulation in a time-dependent manner. In Chapter 5, I further engineered CRISPR single guide RNA (sgRNA) scaffolds by adding aptamer sequences. The RNA binding protein was fused with one of the dimerizers; while the other dimerizer was fused with p65-HSF1. Efficient gene regulations were achieved, and orthogonality of inducible gene regulation was proved. The new systems used only dCas9Sp, which had less limitation to design specific targeting sites. The induction efficiency could potentially be further improved by inserting more copies of aptamer sequences in the future work.
Issue Date:2021-04-22
Rights Information:Copyright 2021 Xiong Xiong
Date Available in IDEALS:2021-09-17
Date Deposited:2021-05

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