{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/108318"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/108318","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Mammalian genome engineering: Fundamental investigation and method development","abstract":"DNA lies at the base of the central dogma of life. Altering DNA enables modification of information flow carried on by fundamental cellular processes like transcription and translation. The ability to precisely manipulate DNA has led to remarkable advances in treating incurable human genetic ailments and has changed the landscape of biological research. Recent decades have ushered in discovery of bacterial proteins that have been adapted for use in mammalian systems. TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are the most widely employed gene-editing tools presently. The modularity and specificity of these gene-editing proteins has made it possible to efficiently engineer mammalian genomes. Understanding the genome-search mechanism of structurally distinct TALEN and CRISPR/Cas9 at the molecular level will not only increase our fundamental understanding of DNA-binding proteins but also enable improvement of these powerful tools. In part 1 of this dissertation (Chapter 2 and 3), I studied the target-search mechanism of TALE and dCas9 proteins in live-mammalian cells using single molecule microscopy. The first part focuses on elucidating TALE search mechanism in vivo. For the first time, TALE genome-search has been visualized in real-time in live-cells and search kinetics were extracted. The results show that TALEs display heterogeneous diffusion behaviors. On further elucidation of the slow diffusion molecules, we discover evidence for local search in the genome. Overall, TALEs undergo facilitated diffusion in the crowded nucleoplasm of a live HeLa cell. In Chapter 3, I performed a comparative single-molecule and editing efficiency analysis to study TALE and CRISPR/Cas9 performance in heterochromatin. Single TALE molecules appear to be less restricted in heterochromatin in comparison to Cas9 molecules. The search mechanism of these proteins correlates with their editing efficiency as TALENs are upto 5-times better than Cas9 in editing target-sites present in compact heterochromatin loci. These findings will govern design rules for developing genome-editing tools for rewiring heterochromatin as well as for biomedical applications. The aim of the second-part of my dissertation (Chapter 4 and 5) is to develop genome editing tools for mammalian genome engineering. Chapter 4 describes a method that I developed to safeguard the most widely used genome-editing technology, CRISPR/Cas9. CRISPR/Cas9 are modular DNA-binding proteins that can recognize a customized DNA-sequence when programmed with a complementary gRNA molecule. The recently discovered of Anti-CRISPR proteins act as a kill switch of Cas9 in nature. I repurposed these proteins to achieve secondary control over Cas9 gene editing activity by combining Anti-CRISPR fusion proteins with a destabilization domain that can be activated with a small molecule. Dose-dependent control over Cas9 editing and dCas9-mediated activation can be achieved. By timely induction of Anti-CRISPR protein expression, off-target activity of Cas9 protein is shown to be reduced significantly. Overall, this toolset is a proof-of-concept for improving Cas9 specificity and biosafety for gene-editing applications. In Chapter 5, I describe my ongoing efforts in developing a multi-factorial Ischemia engineered cell-therapy using CRISPR/Cas9-based gene activation systems. I used synthetic biology tools that can be controlled by chemical ligands to. Temporally induce three important growth factors that control the process of blood-vessel maturation in case of an ischemic injury. Single growth factor therapies have proven to be ineffective in Phase II/III clinical trials. I devised a multi-factorial combinatorial therapy involving neo-angiogenic, anti-inflammatory and anti-angiogenic pathways activated by orthogonal ligands.","abstract_html":"DNA lies at the base of the central dogma of life. Altering DNA enables modification of information flow carried on by fundamental cellular processes like transcription and translation. The ability to precisely manipulate DNA has led to remarkable advances in treating incurable human genetic ailments and has changed the landscape of biological research. Recent decades have ushered in discovery of bacterial proteins that have been adapted for use in mammalian systems. TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are the most widely employed gene-editing tools presently. The modularity and specificity of these gene-editing proteins has made it possible to efficiently engineer mammalian genomes. Understanding the genome-search mechanism of structurally distinct TALEN and CRISPR/Cas9 at the molecular level will not only increase our fundamental understanding of DNA-binding proteins but also enable improvement of these powerful tools. In part 1 of this dissertation (Chapter 2 and 3), I studied the target-search mechanism of TALE and dCas9 proteins in live-mammalian cells using single molecule microscopy. The first part focuses on elucidating TALE search mechanism in vivo. For the first time, TALE genome-search has been visualized in real-time in live-cells and search kinetics were extracted. The results show that TALEs display heterogeneous diffusion behaviors. On further elucidation of the slow diffusion molecules, we discover evidence for local search in the genome. Overall, TALEs undergo facilitated diffusion in the crowded nucleoplasm of a live HeLa cell. In Chapter 3, I performed a comparative single-molecule and editing efficiency analysis to study TALE and CRISPR/Cas9 performance in heterochromatin. Single TALE molecules appear to be less restricted in heterochromatin in comparison to Cas9 molecules. The search mechanism of these proteins correlates with their editing efficiency as TALENs are upto 5-times better than Cas9 in editing target-sites present in compact heterochromatin loci. These findings will govern design rules for developing genome-editing tools for rewiring heterochromatin as well as for biomedical applications. The aim of the second-part of my dissertation (Chapter 4 and 5) is to develop genome editing tools for mammalian genome engineering. Chapter 4 describes a method that I developed to safeguard the most widely used genome-editing technology, CRISPR/Cas9. CRISPR/Cas9 are modular DNA-binding proteins that can recognize a customized DNA-sequence when programmed with a complementary gRNA molecule. The recently discovered of Anti-CRISPR proteins act as a kill switch of Cas9 in nature. I repurposed these proteins to achieve secondary control over Cas9 gene editing activity by combining Anti-CRISPR fusion proteins with a destabilization domain that can be activated with a small molecule. Dose-dependent control over Cas9 editing and dCas9-mediated activation can be achieved. By timely induction of Anti-CRISPR protein expression, off-target activity of Cas9 protein is shown to be reduced significantly. Overall, this toolset is a proof-of-concept for improving Cas9 specificity and biosafety for gene-editing applications. In Chapter 5, I describe my ongoing efforts in developing a multi-factorial Ischemia engineered cell-therapy using CRISPR/Cas9-based gene activation systems. I used synthetic biology tools that can be controlled by chemical ligands to. Temporally induce three important growth factors that control the process of blood-vessel maturation in case of an ischemic injury. Single growth factor therapies have proven to be ineffective in Phase II/III clinical trials. I devised a multi-factorial combinatorial therapy involving neo-angiogenic, anti-inflammatory and anti-angiogenic pathways activated by orthogonal ligands.","abstract_has_math":false,"creators":["Jain, Surbhi"],"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","Shapiro, David R","Schroeder, Charles M","Zhang, Kai"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2020,"date_issued":"2020-08-27T00:51:25Z","date_published":"2020-08-27T00:51:25Z","updated_at":"2026-07-22T22:24:48Z","subjects":["CRISPR/Cas9, TALEN, Genome Engineering, Mammalian Synthetic Biology"],"languages":["en"],"rights":["Copyright 2020 Surbhi Jain"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/108318","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zhao, Huimin","Shapiro, David R","Schroeder, Charles M","Zhang, Kai"]},{"key":"dc:creator","label":"Author","values":["Jain, Surbhi"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2020-08-27T00:51:25Z","2022-08-27T00:51:40Z","2020-05-08","2020-05"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"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":["CRISPR/Cas9, TALEN, Genome Engineering, Mammalian Synthetic Biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2020 Surbhi Jain"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/108318"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["DNA lies at the base of the central dogma of life. Altering DNA enables modification of information flow carried on by fundamental cellular processes like transcription and translation. The ability to precisely manipulate DNA has led to remarkable advances in treating incurable human genetic ailments and has changed the landscape of biological research. Recent decades have ushered in discovery of bacterial proteins that have been adapted for use in mammalian systems. TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are the most widely employed gene-editing tools presently. The modularity and specificity of these gene-editing proteins has made it possible to efficiently engineer mammalian genomes. Understanding the genome-search mechanism of structurally distinct TALEN and CRISPR/Cas9 at the molecular level will not only increase our fundamental understanding of DNA-binding proteins but also enable improvement of these powerful tools. In part 1 of this dissertation (Chapter 2 and 3), I studied the target-search mechanism of TALE and dCas9 proteins in live-mammalian cells using single molecule microscopy. The first part focuses on elucidating TALE search mechanism in vivo. For the first time, TALE genome-search has been visualized in real-time in live-cells and search kinetics were extracted. The results show that TALEs display heterogeneous diffusion behaviors. On further elucidation of the slow diffusion molecules, we discover evidence for local search in the genome. Overall, TALEs undergo facilitated diffusion in the crowded nucleoplasm of a live HeLa cell. In Chapter 3, I performed a comparative single-molecule and editing efficiency analysis to study TALE and CRISPR/Cas9 performance in heterochromatin. Single TALE molecules appear to be less restricted in heterochromatin in comparison to Cas9 molecules. The search mechanism of these proteins correlates with their editing efficiency as TALENs are upto 5-times better than Cas9 in editing target-sites present in compact heterochromatin loci. These findings will govern design rules for developing genome-editing tools for rewiring heterochromatin as well as for biomedical applications. The aim of the second-part of my dissertation (Chapter 4 and 5) is to develop genome editing tools for mammalian genome engineering. Chapter 4 describes a method that I developed to safeguard the most widely used genome-editing technology, CRISPR/Cas9. CRISPR/Cas9 are modular DNA-binding proteins that can recognize a customized DNA-sequence when programmed with a complementary gRNA molecule. The recently discovered of Anti-CRISPR proteins act as a kill switch of Cas9 in nature. I repurposed these proteins to achieve secondary control over Cas9 gene editing activity by combining Anti-CRISPR fusion proteins with a destabilization domain that can be activated with a small molecule. Dose-dependent control over Cas9 editing and dCas9-mediated activation can be achieved. By timely induction of Anti-CRISPR protein expression, off-target activity of Cas9 protein is shown to be reduced significantly. Overall, this toolset is a proof-of-concept for improving Cas9 specificity and biosafety for gene-editing applications. In Chapter 5, I describe my ongoing efforts in developing a multi-factorial Ischemia engineered cell-therapy using CRISPR/Cas9-based gene activation systems. I used synthetic biology tools that can be controlled by chemical ligands to. Temporally induce three important growth factors that control the process of blood-vessel maturation in case of an ischemic injury. Single growth factor therapies have proven to be ineffective in Phase II/III clinical trials. I devised a multi-factorial combinatorial therapy involving neo-angiogenic, anti-inflammatory and anti-angiogenic pathways activated by orthogonal ligands.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2022-05-01","The student, Surbhi Jain, accepted the attached license on 2020-05-05 at 23:51.","The student, Surbhi Jain, submitted this Dissertation for approval on 2020-05-05 at 23:52.","This Dissertation was approved for publication on 2020-05-08 at 07:28.","DSpace SAF Submission Ingestion Package generated from Vireo submission #15237 on 2020-08-25 at 17:43:15","Made available in DSpace on 2020-08-27T00:51:25Z (GMT). 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Altering DNA enables modification of information flow carried on by fundamental cellular processes like transcription and translation. The ability to precisely manipulate DNA has led to remarkable advances in treating incurable human genetic ailments and has changed the landscape of biological research. Recent decades have ushered in discovery of bacterial proteins that have been adapted for use in mammalian systems. TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are the most widely employed gene-editing tools presently. The modularity and specificity of these gene-editing proteins has made it possible to efficiently engineer mammalian genomes. Understanding the genome-search mechanism of structurally distinct TALEN and CRISPR/Cas9 at the molecular level will not only increase our fundamental understanding of DNA-binding proteins but also enable improvement of these powerful tools. In part 1 of this dissertation (Chapter 2 and 3), I studied the target-search mechanism of TALE and dCas9 proteins in live-mammalian cells using single molecule microscopy. The first part focuses on elucidating TALE search mechanism in vivo. For the first time, TALE genome-search has been visualized in real-time in live-cells and search kinetics were extracted. The results show that TALEs display heterogeneous diffusion behaviors. On further elucidation of the slow diffusion molecules, we discover evidence for local search in the genome. Overall, TALEs undergo facilitated diffusion in the crowded nucleoplasm of a live HeLa cell. In Chapter 3, I performed a comparative single-molecule and editing efficiency analysis to study TALE and CRISPR/Cas9 performance in heterochromatin. Single TALE molecules appear to be less restricted in heterochromatin in comparison to Cas9 molecules. The search mechanism of these proteins correlates with their editing efficiency as TALENs are upto 5-times better than Cas9 in editing target-sites present in compact heterochromatin loci. These findings will govern design rules for developing genome-editing tools for rewiring heterochromatin as well as for biomedical applications. The aim of the second-part of my dissertation (Chapter 4 and 5) is to develop genome editing tools for mammalian genome engineering. Chapter 4 describes a method that I developed to safeguard the most widely used genome-editing technology, CRISPR/Cas9. CRISPR/Cas9 are modular DNA-binding proteins that can recognize a customized DNA-sequence when programmed with a complementary gRNA molecule. The recently discovered of Anti-CRISPR proteins act as a kill switch of Cas9 in nature. I repurposed these proteins to achieve secondary control over Cas9 gene editing activity by combining Anti-CRISPR fusion proteins with a destabilization domain that can be activated with a small molecule. Dose-dependent control over Cas9 editing and dCas9-mediated activation can be achieved. By timely induction of Anti-CRISPR protein expression, off-target activity of Cas9 protein is shown to be reduced significantly. Overall, this toolset is a proof-of-concept for improving Cas9 specificity and biosafety for gene-editing applications. In Chapter 5, I describe my ongoing efforts in developing a multi-factorial Ischemia engineered cell-therapy using CRISPR/Cas9-based gene activation systems. I used synthetic biology tools that can be controlled by chemical ligands to. Temporally induce three important growth factors that control the process of blood-vessel maturation in case of an ischemic injury. Single growth factor therapies have proven to be ineffective in Phase II/III clinical trials. I devised a multi-factorial combinatorial therapy involving neo-angiogenic, anti-inflammatory and anti-angiogenic pathways activated by orthogonal ligands.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2022-05-01","The student, Surbhi Jain, accepted the attached license on 2020-05-05 at 23:51.","The student, Surbhi Jain, submitted this Dissertation for approval on 2020-05-05 at 23:52.","This Dissertation was approved for publication on 2020-05-08 at 07:28.","DSpace SAF Submission Ingestion Package generated from Vireo submission #15237 on 2020-08-25 at 17:43:15","Made available in DSpace on 2020-08-27T00:51:25Z (GMT). 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