CRISPR-based genome editing tools for virus resistance in grapevine
Date
2022-12
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Grapevine (Vitis vinifera) is an important fruit crop which contributes significantly to the South
African agricultural sector, and is a major produce crop worldwide. Grapevine viruses are
widespread and cause serious diseases which impact the quality and quantity of crop yields. More
than 80 viruses plague grapevine, with RNA viruses constituting the largest of virus pathogens.
Clustered regularly interspaced, short palindromic repeat (CRISPR), along with its CRISPR-
associated (Cas) proteins, is a system which has been harnessed from the prokaryotic immune system
and adapted for genome editing technologies. The first CRISPR system to be adapted for genome
editing was CRISPR/Cas9, which is characterised by its ability to target double-strand DNA. A
recent extension to the CRISPR armoury is the Cas13 effector, which exclusively targets
single-strand RNA. CRISPR/Cas has been implemented as a defence mechanism in plants, against both
DNA and RNA viruses, by being programmed to directly target and cleave the viral genomes. The
efficacy of the CRISPR/Cas tool in plants is dependent on efficient delivery of its components into
plant cells. Geminiviruses, a group of small DNA viruses with a useful replication mechanism, have
been reconstructed into efficient expression vectors and used for the delivery of genome editing
components. By harnessing the CRISPR/Cas tool, and implementing the use of a viral vector for the
expression thereof, a robust approach to induce virus resistance in plants can be achieved. To this
end, the first aim of this study was to use CRISPR/CasRx to target an infectious clone of the RNA
virus, grapevine virus A (GVA). GVA naturally infects V. vinifera, but can infect the model plant
Nicotiana benthamiana, making it a helpful model to study virus infection in grapevine. The second
aim of this study sought to use a geminivirus vector based on the bean yellow dwarf virus (BeYDV)
to deliver and express CRISPR/Cas9 components in N. benthamiana. Firstly, constructs harbouring
CasRx and a guide RNA (gRNA) targeting the replicase gene of GVA were assembled, and used for
Agrobacterium-mediated transformation of N. benthamiana. Transgenic lines were infiltrated with the
GVA infectious clone, but no consistent GVA interference was observed. To improve virus targeting,
gRNAs were designed against the coat protein (CP) gene of GVA. N. benthamiana plants expressing
CasRx were co-infiltrated with the infectious clone, and with a tobacco rattle virus (TRV)-gRNA
expression vector, harbouring a CP gRNA. Results indicated more consistent GVA reductions,
specifically CP gRNA 2, which demonstrated a significant negative correlation with GVA
accumulation, as well as multiple gRNA co-infiltrations which similarly showed reduced GVA titre.
When the pRIC BeYDV vector was used
for gene targeting with CRISPR/Cas9, exogenously-delivered enhanced green fluorescence
protein (eGFP), as well as endogenous N. benthamiana genes phytoene desaturase (PDS), and green
fluorescence protein (GFP), from the transgenic N. benthamiana 16c line, were successfully cleaved
and edited. By establishing a virus-targeting defence system in plants, and utilising a high-
expressing geminivirus vector for the delivery of genome editing components, efficient virus
interference mechanisms can be established and applied to major crops, such as grapevine.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.
Description
Thesis (MScAgric)--Stellenbosch University, 2022.
Keywords
CRISPR (Genetics), Genome editing, Grapevine (Vitis vinifera) -- Virus diseases, Plants -- Virus resistance, CRISPR/Cas, CRISPR-associated protein 9, UCTD