Masters Degrees (Genetics)

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CRISPR-based genome editing tools for virus resistance in grapevine
(Stellenbosch : Stellenbosch University, 2022-12) Spencer, Katarina Paula; Burger, Johan; Campa, Manuela; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
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.
Establishing 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.
Genome editing in bread wheat using CRISPR/Cas9
(Stellenbosch : Stellenbosch University, 2022-04) Dijkerman, Alexander; Burger, Johan; Campa, Manuela; Botes, Willem; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
ENGLISH ABSTRACT: Bread wheat (Triticum aestivum) is one of the most important food crops consumed by humans, providing approximately 20% of the world’s total caloric intake. However, wheat yields must be increased to supply the growing demand of an increasing global population. Traditional breeding techniques will not be sufficient to confront this challenge and improved genetic engineering and molecular-based techniques will be a necessity. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated) technology has emerged as a promising genetic engineering tool for the purposes of plant breeding. The CRISPR/Cas9 system has recently been developed as a DNA-free genome editing technique allowing for a precise and efficient method to genetically improve bread wheat while mitigating regulatory concerns. This study, therefore, aimed to establish CRISPR/Cas9 system in bread wheat and investigate the feasibility of this system in a DNA-free format. To achieve this, ribonucleoproteins (RNPs) were assembled by complexing single guide RNA (sgRNA) sequences targeting regions of a gene involved in the carotenoid pathway to the Cas9 protein. The RNPs were subsequently introduced to immature embryos through biolistic bombardment. Immature embryos were assessed to confirm successful editing of the target genes. No editing was detected in the experimental target. Evidence is provided for successful editing in embryos bombarded with RNPs mediated by a previously validated sgRNA. To establish an efficient method of achieving CRISPR/Cas9 edited wheat plants, a multiplex CRISPR/Cas9 DNA construct was assembled and introduced into wheat tissues through Agrobacterium-mediated transformation and particle bombardment. No edits were detected in plantlets that were regenerated on selective media from embryos transformed with Agrobacterium carrying the CRISPR/Cas9 construct. However, indels were detected in pre-initiated calli bombarded with the multiplex CRISPR/Cas9 DNA construct when analysed with the ICE v2 online software. Furthermore, various wheat transformation and regeneration protocols are assessed. Overall, the results provide insights into methods to deliver CRISPR/Cas9 components into bread wheat explant tissue for genome editing. A rapid and accessible method of screening for edits in pooled samples through PCR/RE assays followed by ICE v2 software analyses is demonstrated. Further, a vector delivery method that could circumvent challenging tissue culture procedures through the bombardment of mature embryos is explored. Possible optimisations of CRISPR/Cas delivery systems and experimental design are highlighted for future studies.
Investigation of the DMR6 susceptibility genes in grapevine for improving phytoplasma resistance through CRISPR/Cas9 technology
(Stellenbosch : Stellenbosch University, 2024-03) Holm, Clara Cornelia; Burger, Johan; Campa, Manuela; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: Vi6culture is considered one of the most important crop industries worldwide but is con6nually placed under pressure from a vast array of pathogens. Phytoplasma diseases are a cri6cal challenge for the grapevine industry and cause considerable yield losses. Several breeding strategies aim to improve grapevine disease resistance through the introgression of resistance genes, or through the inac6va6on of suscep6bility (S) genes, using genome edi6ng. Recent advancements of genome edi6ng tools such as CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) allows for the targeted inac6va6on of important host S factors that play a role during pathogen infec6on. The Downy Mildew Resistant 6 (DMR6) genes were ini6ally described in Arabidopsis thaliana as a suscep6bility factor for bacterial and oomycete pathogens. Subsequent inac6va6on of these genes resulted in broad range pathogen resistance in various crops such as tomato, leBuce, cucumber, pepper, and citrus. Furthermore, there is evidence that the VviDMR6 genes play a similar role in grapevine. In fact, most recently the inac6va6on of the VviDMR6 genes lead to enhanced downy mildew resistance in grapevine. However, it’s precise role during pathogenesis remains unclear. To this end, this study aimed to func6onally characterize the grapevine DMR6 genes, with par6cular focus on their involvement in plant defense. Gene co- expression networks highlighted the involvement of VviDMR6.1 in defense, while VviDMR6.2 was associated with developmental pathways. Gene expression analyses across diﬀerent grapevine 6ssues revealed that VviDMR6.1 was predominantly expressed in the roots. Furthermore, to beBer understand the role of VviDMR6 during pathogenesis, in vitro grown grapevine was treated with salicylic acid (SA) to examine its response in diﬀerent 6ssue types. Both VviDMR6 genes were induced upon SA treatment across diﬀerent 6ssues. Addi6onally, GUS-reporter assays were used to further inves6gate the 6ssue speciﬁcity of the VviDMR6 genes. This entailed the iden6ﬁca6on and isola6on of the puta6ve promoter regions of VviDMR6.1 and VviDMR6.2 from Chardonnay. In silico analyses unveiled core promoter elements and puta6ve regulatory mo6fs associated with stress responsiveness within both VviDMR6 promoters. Subsequently, two promoterDMR6::GUS vectors were constructed consis6ng of the respec6ve promoters fused to GUS, and were subsequently introduced into A. thaliana. The proDMR6.1::GUS transgenic lines showed dis6nct promoter ac6vity in the roots of fully grown plants and demonstrated clear induc6on upon SA exposure. Finally, two previously designed CRISPR/Cas9 constructs containing two targets for VviDMR6.1 and a single target for VviDMR6.2 were introduced to rootstock cul6var Richter 110 embryogenic callus through Agrobacterium-mediated transforma6on. Successful edi6ng of VviDMR6.1 was achieved, with a muta6on frequency of 43%. Inference of CRISPR Edits (ICE) analysis revealed small indels, primarily 2-bp dele6ons, in the VviDMR6.1 edited lines. Notably, a single transgenic line demonstrated complete edi6ng, achieving 100% edi6ng eﬃciency. This contributes to the advancement of genome edi6ng tools for enhancing disease resistance in grapevine and yielded the ﬁrst CRISPR/Cas9-edited grapevine on the African con6nent.
An investigation of the potato eIF4E isoforms as targets for non-transgenic CRISPR/Cas9 genome editing for viral resistance
(Stellenbosch : Stellenbosch University, 2022-12) Hurst, Rebecca; Lloyd, James Richard; Burger, Johan; Campa, Manuela; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
ENGLISH ABSTRACT: Solanum tuberosum (potato) is an important food source in Southern Africa. Viral infection of potato plants leads to decreased tuber size and number, and symptomatic tubers are often unfit for consumption. Increased prevalence of the viruses Potato Virus Y (PVY) and Potato Leaf Roll Virus (PLRV) in South Africa threatens the country’s food security. The eukaryotic translation initiation factor 4E (eIF4E) gene family encode proteins that are involved in native and viral RNA translation. This mechanism is essential for viral survival and the eIF4E family are promising susceptibility factors that can be manipulated to confer viral resistance in plants. Expression of the potato eIF4E isoforms – eIF4E-1, eIF4E-2, eIF(iso)4E – and the related gene new RNA cap binding protein (nCBP), was investigated in tubers of PLRV-infected plants and compared to that of tubers from healthy plants. No significant difference was observed between the samples which may indicate that expression differences are tissue-specific, rather than stress-induced. A Bayesian maximum clade credibility tree was created to elucidate the emergence of eIF4E isoforms across plant evolution. Division of the plant RNA cap-binding proteins into two distinct groups - eIF4E and nCBP –occurred in the common ancestor of all land plants. The eIF4E-ancestor divided into eIF4E and eIF(iso)4E in at least the angiosperms and possibly as far back as the vascular plants. The further division of eIF4E into eIF4E-1 and eIF4E-2 only occurred recently, in an ancestor of the Solanaceous family. During infection, the essential interaction between the viral genome-linked protein (VPg) and the host translation machinery is facilitated by three amino acid residues in the eIF4E cap-binding pocket. Amino acid alignments of the VPg binding region of the potato eIF4E proteins indicate that all have the potential for interaction with the viral protein. This implies that the VPg binding capacity of all these genes would need to be disrupted to engineer complete resistance to PVY and PLRV. To begin the process of creating eIF4E-1, eIF4E-2 and eIF(iso)4E knockout mutants in potato, single guide RNAs were designed for all three genes. Additionally, a single guide RNA that is capable of simultaneously knocking out eIF4E-1 and eIF4E-2 was designed. Each of these guides were transcribed in vitro and complexed with Cas9. In vitro efficacy assays demonstrated that all ribonucleoproteins could induce double stranded breaks to the target genes. Transformation of the CRISPR/Cas9 ribonucleoproteins into protoplasts provides a transgene-free method of eIF4E gene editing. The isolation of viable potato protoplasts was established, and tissue culture of these cells yielded micro-calli.

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Browsing Masters Degrees (Genetics) by browse.metadata.advisor "Burger, Johan"

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