Browsing by Author "Robertson, Gaëlle"
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- ItemEstablishing the CRISPR/Cas13a genome editing system in Nicotiana benthamiana for RNA targeting applications(Stellenbosch : Stellenbosch University, 2021-12) Robertson, Gaëlle; Burger, Johan; Campa, Manuela; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Globally, grapevine (Vitis vinifera) is a widely cultivated fruit crop and makes a vital contribution to the South African agricultural sector. Highly susceptible to a plethora of virus species, grapevine faces severe constraints to overall crop productivity and durable antiviral strategies are necessary to control the spread of viral diseases. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated) technology has emerged as a valuable genetic engineering tool for plant breeding purposes. Recently, genome editing by the CRISPR/Cas system has been expanded beyond DNA targeting. A novel class 2 Cas effector, Cas13a, has been revealed as a programmable RNA- targeting nuclease. Using CRISPR/Cas13a, this study therefore aimed to investigate the potential of this system to mediate targeted RNA cleavage and viral RNA interference in Nicotiana benthamiana. The choice of this model plant allowed for an immediate test of the functionality and efficacy of this system. For this, binary Cas13a vectors targeting regions of an annotated mRNA transcript from the carotenoid pathway were assembled and transgenic N. benthamiana lines were established. A CRISPR/Cas13a-based down-regulation of gene expression was not observed in these lines. However, after improving the cellular localisation of the Cas13a/crRNA constructs, a transgenic line expressing a cytoplasmic-localised Cas13a/crRNA vector showed a significant two-fold reduction in target gene expression, further correlated with a lowered concentration of total carotenoid content from a preliminary measurement. To demonstrate virus inhibition, a modified tobacco mosaic virus (TMV) system expressing a green fluorescent protein (TRBO-GFP), was used to visually and molecularly measure CRISPR/Cas13a-mediated interference activity in N. benthamiana. A LwaCas13a/crRNA vector targeting a conserved region of the reporter mRNA was assembled and after performing a series of transient assays, phenotypic quantifications of GFP signal intensity confirmed a significant attenuation (~50%) in virus accumulation. However, RT-qPCR analyses showed that GFP mRNA abundance was not directly proportional to that of the observed GFP signal intensity, suggesting a possible limitation in the method of molecular quantification of the GFP mRNA transcript levels. Overall, the results provide insight into the functionality of the CRISPR/Cas13a system for RNA targeting of both an endogenous transcript and a viral genome in plants. Further optimisation of crRNA design features and the method of CRISPR component delivery are highlighted for future studies, especially for an application in grapevine.