Exploring Non-transgenic CRISPR-Cas9 gRNA Delivery Using Transactivation in Nicotiana benthamiana
Gene editing via CRISPR-Cas9 (Clustered, Regularly Interspaced, Short Palindromic Repeats – CRISPR-associated protein 9) is a powerful tool in biotechnology. The method involves an endonuclease Cas9 forming a complex with a guide RNA (gRNA) that matches a distinct DNA target sequence, causing double-stranded breaks in the DNA site. The break induces imperfect DNA repair resulting in insertions or deletions that render the target gene non-functional. It is also possible to mutate the Cas9 protein so that it loses its cutting function, but can still sit on the target DNA when in a complex with gRNA. This deactivated Cas9 (dCas9) can be fused with transcriptional activators or repressors to adjust the expression of a target gene. While CRISPR-Cas9 is already widely used in plants, there are limitations to the system’s delivery and efficiency. The aims of the research in this thesis were to generate a dCas9-activator-reporter system and to use the system to explore the possibility of two different non-transgenic methods of delivering gRNA. The system consists of two plant expression plasmids. One encodes a constitutively expressed dCas9 fused to a transcriptional activator and a reporter gene driven by a minimal promoter. The second contains the gRNA, designed to target the minimal promoter. When the constructs are co-expressed, the gRNA-bound dCas9-activator sits on the minimal promoter and drives increased reporter expression. Transient assays in N. benthamiana established the functionality of the system, showing increased reporter levels when the two constructs were co-expressed compared to the activator- reporter construct alone. Stable transgenic N. benthamiana lines of the separate constructs were generated and taken to the T2 generation. To explore non-transgenic gRNA introduction methods, I tested viral delivery and grafting. Tobacco Rattle Virus (TRV) engineered to contain gRNA for the system was able to activate the reporter. However, the results suggest that viral recombination of the gRNA insert may cause the effect to be lost over time. The positive readout transactivation system developed in this thesis will be a valuable tool for future CRISPR development in plants. In addition, the data reported present opportunities for further exploration on potential delivery methods, spatial specificity of CRISPR, and the mobility characteristics of synthetic gRNA compared to various endogenous RNAs.
Networked Digital Library of Theses and Dissertations