Assessment and Characterization of Various Gene-editing Platforms for Brassica napus (Canola) using TRANSPARENT TESTA 8 (TT8) as the Target Gene

Coates, Ethan Robin

Assessment and Characterization of Various Gene-editing Platforms for Brassica napus (Canola) using TRANSPARENT TESTA 8 (TT8) as the Target Gene

dc.contributor.advisor	Samuel, Marcus
dc.contributor.author	Coates, Ethan Robin
dc.contributor.committeemember	Muench, Douglas
dc.contributor.committeemember	Ro, Dae-Kyun
dc.contributor.committeemember	Chua, Gordon
dc.date	2024-02
dc.date.accessioned	2024-01-03T16:50:53Z
dc.date.available	2024-01-03T16:50:53Z
dc.date.issued	2023-12-21
dc.description.abstract	Climate change poses a significant threat to agriculture by causing more frequent and extreme weather events, altering temperature and precipitation patterns, and increasing the prevalence of pests and diseases. These changes can lead to reduced crop yields and jeopardize food security on a global scale. CRISPR-Cas9, a revolutionary gene editing tool, offers a promising solution to address these challenges by enabling the development of crops with enhanced resilience to environmental stressors. Through targeted genetic modifications, it is possible to create crop varieties that are more drought-tolerant, heat-resistant, and better equipped to adapt to changing conditions, ultimately ensuring a more sustainable and secure food supply in the face of climate change. However, a multitude of different CRISPR systems with varying success, contingent on factors such as the plant species, sgRNA expression, and the choice of endonuclease present unique considerations. For instance, the choice between Cas9 versus an alternative, and the selection between a polycistronic or single guide complex system, can impact outcomes. In this thesis, I aimed to assess efficiency of several CRISPR systems in Brassica napus, a globally important oilseed crop also known as canola, in order to identify the most efficient editing system for canola. By investigating sgRNA expression and endonuclease efficiency using a hairy root system, the findings revealed that the 35S:Cas9:m35S:tRNA guide system was the most effective for editing the target gene, the transcription factor TT8 (Transparent Testa 8), in B. napus hairy roots. The choice of the 35S promoter driving the Cas9 and sgRNA expression in the 35S:Cas9:m35S:tRNA guide system appeared to contribute to its success, allowing for simultaneous and stoichiometric availability of Cas9 protein and its target guide RNA. This system can be efficiently utilized in future stable of hairy root systems to target candidate genes.
dc.identifier.citation	Coates, E. R. (2023). Assessment and characterization of various gene-editing platforms for Brassica napus (Canola) using Transparent Testa 8 (TT8) as the target gene (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.
dc.identifier.uri	https://hdl.handle.net/1880/117833
dc.identifier.uri	https://doi.org/10.11575/PRISM/42676
dc.language.iso	en
dc.publisher.faculty	Graduate Studies
dc.publisher.institution	University of Calgary
dc.rights	University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subject	Gene editing
dc.subject	Canola
dc.subject.classification	Biology--Molecular
dc.subject.classification	Agriculture
dc.title	Assessment and Characterization of Various Gene-editing Platforms for Brassica napus (Canola) using TRANSPARENT TESTA 8 (TT8) as the Target Gene
dc.type	master thesis
thesis.degree.discipline	Biological Sciences
thesis.degree.grantor	University of Calgary
thesis.degree.name	Master of Science (MSc)
ucalgary.thesis.accesssetbystudent	I do not require a thesis withhold – my thesis will have open access and can be viewed and downloaded publicly as soon as possible.