{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/97547"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/97547","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Multiplex genome editing and gene regulation in eukaryotic cells","abstract":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-05-01","abstract_html":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-05-01","abstract_has_math":false,"creators":["Bao, Zehua"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Zhao, Huimin","Morrissey, James H.","Chen, Jie","Zhang, Kai"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017-08-10T19:51:45Z","date_published":"2017-08-10T19:51:45Z","updated_at":"2026-07-22T22:24:34Z","subjects":["Genome editing","Genome engineering","Gene regulation","Clustered, regularly interspaced short palindromic repeats (CRISPR)","Cas9","Saccharomyces cerevisiae"],"languages":["en"],"rights":["Copyright 2017 Zehua Bao"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/97547","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zhao, Huimin","Morrissey, James H.","Chen, Jie","Zhang, Kai"]},{"key":"dc:creator","label":"Author","values":["Bao, Zehua"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2017-08-10T19:51:45Z","2019-08-11T09:15:21Z","2017-04-10","2017-05"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Genome editing","Genome engineering","Gene regulation","Clustered, regularly interspaced short palindromic repeats (CRISPR)","Cas9","Saccharomyces cerevisiae"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2017 Zehua Bao"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/97547"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-05-01","The student, Zehua Bao, accepted the attached license on 2017-04-05 at 11:57.","The student, Zehua Bao, submitted this Dissertation for approval on 2017-04-05 at 12:29.","Biological functions of a cell are encoded within its genome and regulated through complex genetic networks. Direct manipulation of the genome and the transcriptome in multiplex enables study of gene-gene interactions and rapid creation of industrially or therapeutically useful cells. Such manipulations, including genome editing and epigenetic regulation of gene expression, rely on a group of precise DNA-binding proteins, which include zinc finger proteins (ZFPs), transcription activator like effectors (TALEs), and clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9). ZFPs and TALEs rely on protein domain shuffling for targeting different genomic loci, which can be laborious and expensive. Instead, CRISPR/Cas9 is guided by a 20 bp ribonucleotide, which can be synthesized in multiplex rapidly and cost-effectively. This dissertation describes my efforts in engineering the CRISPR/Cas9 system for multiplex genome editing and gene regulation in eukaryotic cells. The first part of this dissertation (Chapters 2 and 3) focuses on genome editing in Saccharomyces cerevisiae. S. cerevisiae, also known as the baker’s yeast, is an important model eukaryotic organism for basic research, as well as a preferred industrial host for production of biofuels, pharmaceuticals and fine chemicals. I developed a Homology-Integrated CRISPR/Cas9 (HI-CRISPR) system for concurrent disruption of up to three genes in S. cerevisiae. This system enabled rapid and efficient generation of yeast strains with multiple mutations and should prove useful for studying yeast gene functions. Based on the design of the HI-CRISPR system, I further developed a CRISPR/Cas9 and homology-directed repair assisted genome-scale engineering (CHAnGE) system for engineering and directed evolution of S. cerevisiae strains. Using the CHAnGE system, I identified both known and novel genes susceptible to acetic acid or furfural toxicity. Finally, I demonstrated one-step, high efficiency in vivo site directed mutagenesis in S. cerevisiae for introducing designed mutations into native chromosomal contexts. The second part of this dissertation (Chapters 4 and 5) focuses on ligand-inducible, multiplex gene regulation in mammalian cells. Ligand-inducible gene regulation enables temporal control of gene functions, which is necessary in interrogating dynamic gene regulatory networks. However, temporal control is currently limited on a single gene level. I developed chemically induced transcription activators by combining orthogonal CRISPR/Cas9 systems and chemically induced dimerizing proteins. In HEK293T cells, these transcription activators exerted simultaneous activation of multiple genes and orthogonal regulation of different genes in a ligand-dependent manner with minimal background. As proof of concept, I attempted to convert mouse embryonic fibroblasts to neuronal cells using chemically induced CRISPR/Cas9 activators.","This Dissertation was approved for publication on 2017-04-10 at 16:47.","DSpace SAF Submission Ingestion Package generated from Vireo submission #10651 on 2017-08-10 at 14:30:07","Made available in DSpace on 2017-08-10T19:51:45Z (GMT). No. of bitstreams: 2 BAO-DISSERTATION-2017.pdf: 16612468 bytes, checksum: 6fc970c9543fe94785fb80ad4a730060 (MD5) LICENSE.txt: 4206 bytes, checksum: b0a140e48686298e97b352bff5fcb9b2 (MD5) Previous issue date: 2017-04-10","Embargo set by: Colleen Fallaw for item 102600 Lift date: 2019-08-10T21:25:30Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 102600 on 2019-08-11T09:15:21Z."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Multiplex genome editing and gene regulation in eukaryotic cells"]}]}],"canonical_facts":{"dc:contributor":["Zhao, Huimin","Morrissey, James H.","Chen, Jie","Zhang, Kai"],"dc:creator":["Bao, Zehua"],"dc:date":["2017-08-10T19:51:45Z","2019-08-11T09:15:21Z","2017-04-10","2017-05"],"dc:description":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-05-01","The student, Zehua Bao, accepted the attached license on 2017-04-05 at 11:57.","The student, Zehua Bao, submitted this Dissertation for approval on 2017-04-05 at 12:29.","Biological functions of a cell are encoded within its genome and regulated through complex genetic networks. Direct manipulation of the genome and the transcriptome in multiplex enables study of gene-gene interactions and rapid creation of industrially or therapeutically useful cells. Such manipulations, including genome editing and epigenetic regulation of gene expression, rely on a group of precise DNA-binding proteins, which include zinc finger proteins (ZFPs), transcription activator like effectors (TALEs), and clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9). ZFPs and TALEs rely on protein domain shuffling for targeting different genomic loci, which can be laborious and expensive. Instead, CRISPR/Cas9 is guided by a 20 bp ribonucleotide, which can be synthesized in multiplex rapidly and cost-effectively. This dissertation describes my efforts in engineering the CRISPR/Cas9 system for multiplex genome editing and gene regulation in eukaryotic cells. The first part of this dissertation (Chapters 2 and 3) focuses on genome editing in Saccharomyces cerevisiae. S. cerevisiae, also known as the baker’s yeast, is an important model eukaryotic organism for basic research, as well as a preferred industrial host for production of biofuels, pharmaceuticals and fine chemicals. I developed a Homology-Integrated CRISPR/Cas9 (HI-CRISPR) system for concurrent disruption of up to three genes in S. cerevisiae. This system enabled rapid and efficient generation of yeast strains with multiple mutations and should prove useful for studying yeast gene functions. Based on the design of the HI-CRISPR system, I further developed a CRISPR/Cas9 and homology-directed repair assisted genome-scale engineering (CHAnGE) system for engineering and directed evolution of S. cerevisiae strains. Using the CHAnGE system, I identified both known and novel genes susceptible to acetic acid or furfural toxicity. Finally, I demonstrated one-step, high efficiency in vivo site directed mutagenesis in S. cerevisiae for introducing designed mutations into native chromosomal contexts. The second part of this dissertation (Chapters 4 and 5) focuses on ligand-inducible, multiplex gene regulation in mammalian cells. Ligand-inducible gene regulation enables temporal control of gene functions, which is necessary in interrogating dynamic gene regulatory networks. However, temporal control is currently limited on a single gene level. I developed chemically induced transcription activators by combining orthogonal CRISPR/Cas9 systems and chemically induced dimerizing proteins. In HEK293T cells, these transcription activators exerted simultaneous activation of multiple genes and orthogonal regulation of different genes in a ligand-dependent manner with minimal background. As proof of concept, I attempted to convert mouse embryonic fibroblasts to neuronal cells using chemically induced CRISPR/Cas9 activators.","This Dissertation was approved for publication on 2017-04-10 at 16:47.","DSpace SAF Submission Ingestion Package generated from Vireo submission #10651 on 2017-08-10 at 14:30:07","Made available in DSpace on 2017-08-10T19:51:45Z (GMT). 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