{"id":{"repo_id":"uwo","oai_identifier":"oai:uwo.scholaris.ca:20.500.14721/37274"},"canonical_url":"https://search.dev.ndltd.org/etd/uwo/oai:uwo.scholaris.ca:20.500.14721/37274","repository":{"repo_id":"uwo","name":"Western University","base_url":"https://uwo.scholaris.ca/server/oai/request"},"display":{"title":"Mechanism of Permissive Cleavage Activity of TevCas12a","abstract":"We created a novel dual endonuclease called TevCas12a. TevCas12a is a fusion of the I-TevI nuclease and linker domains to Cas12a. The goal was to create a dual endonuclease that made precise double-strand breaks leaving non-compatible ends for exogenous DNA insertion and specific deletions. TevCas12a was expected to make two double-strand breaks, one by I-TevI and the other from Cas12a. However, when tested in vitro, TevCas12a cut the DNA substrate multiple times, rapidly degrading the substrate. We call this activity permissive cleavage. TevCas12a activity is guide RNA dependent, but not guide RNA sequence specific, it can degrade any DNA substrate with a non-targeted guide RNA. Sequencing of the TevCas12a cleavage products with Oxford Nanopore revealed that most of the non-guide directed cuts were within I-TevI’s cleavage motif, indicating that I-TevI is responsible for the cuts. A mechanism is proposed for how I-TevI and Cas12a work together to permissively cleave DNA.","abstract_html":"We created a novel dual endonuclease called TevCas12a. TevCas12a is a fusion of the I-TevI nuclease and linker domains to Cas12a. The goal was to create a dual endonuclease that made precise double-strand breaks leaving non-compatible ends for exogenous DNA insertion and specific deletions. TevCas12a was expected to make two double-strand breaks, one by I-TevI and the other from Cas12a. However, when tested in vitro, TevCas12a cut the DNA substrate multiple times, rapidly degrading the substrate. We call this activity permissive cleavage. TevCas12a activity is guide RNA dependent, but not guide RNA sequence specific, it can degrade any DNA substrate with a non-targeted guide RNA. Sequencing of the TevCas12a cleavage products with Oxford Nanopore revealed that most of the non-guide directed cuts were within I-TevI’s cleavage motif, indicating that I-TevI is responsible for the cuts. A mechanism is proposed for how I-TevI and Cas12a work together to permissively cleave DNA.","abstract_has_math":false,"creators":["White, Alexa"],"institution":"The University of Western Ontario","degree_name":"M Sc","degree_level":null,"degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":[],"advisors":["Edgell, David R."],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-09-25","date_published":"2023-09-25","updated_at":"2026-07-27T21:55:54Z","subjects":["I-TevI","Cas12a","permissive cleavage","dual endonuclease"],"languages":["en_ca"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/20.500.14721/37274","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Edgell, David R."]},{"key":"dc:creator","label":"Author","values":["White, Alexa"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-07-10T21:32:56Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-07-10T21:32:56Z"]},{"key":"dc:date.issued","label":"Date","values":["2023-09-25"]},{"key":"dc:publisher","label":"Institution","values":["The University of Western Ontario"]},{"key":"dc:type","label":"Dc Type","values":["thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_name","label":"Degree Name","values":["M Sc"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["I-TevI","Cas12a","permissive cleavage","dual endonuclease"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en_ca"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/20.500.14721/37274"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The thesis cover page in the PDF document includes references to Western University’s previous institutional repository platform, known as Scholarship@Western, and links to that platform (beginning with ir.lib.uwo.ca). In citing or referring to this thesis, use the DOI or handle from this page instead. Sample citation: Author name, \"Thesis title.\" (Year). Western University Open Repository. https://doi.org/10.71858/123456."]},{"key":"dc:description.abstract","label":"Abstract","values":["We created a novel dual endonuclease called TevCas12a. TevCas12a is a fusion of the I-TevI nuclease and linker domains to Cas12a. The goal was to create a dual endonuclease that made precise double-strand breaks leaving non-compatible ends for exogenous DNA insertion and specific deletions. TevCas12a was expected to make two double-strand breaks, one by I-TevI and the other from Cas12a. However, when tested in vitro, TevCas12a cut the DNA substrate multiple times, rapidly degrading the substrate. We call this activity permissive cleavage. TevCas12a activity is guide RNA dependent, but not guide RNA sequence specific, it can degrade any DNA substrate with a non-targeted guide RNA. Sequencing of the TevCas12a cleavage products with Oxford Nanopore revealed that most of the non-guide directed cuts were within I-TevI’s cleavage motif, indicating that I-TevI is responsible for the cuts. A mechanism is proposed for how I-TevI and Cas12a work together to permissively cleave DNA."]},{"key":"dc:title","label":"Title","values":["Mechanism of Permissive Cleavage Activity of TevCas12a"]}]}],"canonical_facts":{"dc:contributor.advisor":["Edgell, David R."],"dc:creator":["White, Alexa"],"dc:date.accessioned":["2025-07-10T21:32:56Z"],"dc:date.available":["2025-07-10T21:32:56Z"],"dc:date.issued":["2023-09-25"],"dc:description":["The thesis cover page in the PDF document includes references to Western University’s previous institutional repository platform, known as Scholarship@Western, and links to that platform (beginning with ir.lib.uwo.ca). In citing or referring to this thesis, use the DOI or handle from this page instead. Sample citation: Author name, \"Thesis title.\" (Year). Western University Open Repository. https://doi.org/10.71858/123456."],"dc:description.abstract":["We created a novel dual endonuclease called TevCas12a. TevCas12a is a fusion of the I-TevI nuclease and linker domains to Cas12a. The goal was to create a dual endonuclease that made precise double-strand breaks leaving non-compatible ends for exogenous DNA insertion and specific deletions. TevCas12a was expected to make two double-strand breaks, one by I-TevI and the other from Cas12a. However, when tested in vitro, TevCas12a cut the DNA substrate multiple times, rapidly degrading the substrate. We call this activity permissive cleavage. TevCas12a activity is guide RNA dependent, but not guide RNA sequence specific, it can degrade any DNA substrate with a non-targeted guide RNA. Sequencing of the TevCas12a cleavage products with Oxford Nanopore revealed that most of the non-guide directed cuts were within I-TevI’s cleavage motif, indicating that I-TevI is responsible for the cuts. A mechanism is proposed for how I-TevI and Cas12a work together to permissively cleave DNA."],"dc:identifier.uri":["https://hdl.handle.net/20.500.14721/37274"],"dc:language.iso":["en_ca"],"dc:publisher":["The University of Western Ontario"],"dc:subject":["I-TevI","Cas12a","permissive cleavage","dual endonuclease"],"dc:title":["Mechanism of Permissive Cleavage Activity of TevCas12a"],"dc:type":["thesis"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_name":["M Sc"]},"updated_at":"2026-07-27T21:55:54Z"}