Project Number:
FShip GenUS 17-02
Project title:
CRISPR-1: Grapevine genome editing: development of tools and implementation of the technology
Project leaders:
Vivier, M & Burger, J
Institutions:
South African Grape and Wine Research Institute, Department of Viticulture and Oenology, Stellenbosch University and Department of Genetics, Stellenbosch University
Team members:
Young, P R
Korkie, M
Start date:
2017
Co-funding:
Project is co-funded by the Department of Science and Innovation
EXECUTIVE SUMMARY
Objectives and Rationale
The immense potential of CRISPR/Cas9 technology as a genome editing tool suggests that it can be used in grapevine improvement strategies. This project aimed to establish CRISPR technology in grapevine. We established conventional CRISPR/Cas-based protocols for editing the grapevine genome by targeting a single-copy carotenoid gene at both the genome (DNA) and transcript (RNA) levels. The project comprises four major objectives:
1. Construction of appropriate Cas9- and Cas13a-based editing vectors.
2. Preparation of plant materials for genetic transformation of Nicotiana benthamiana and grapevine.
3. Transformation of N. benthamiana and grapevine and regeneration of plantlets.
4. Evaluation of transformants, both phenotypically, and at a molecular level.
The reason for using N. benthamiana in parallel with grapevine is that it is a fast-growing herbaceous plant that is genetically well-characterised, and amenable to genetic transformation. Moreover, the gene selected for optimising the genome editing system in grapevine, is identical between the two species, suggesting that the editing constructs can be shared, thus saving time and money. Successful achievement of this goal would be an excellent justification to take this project to the next level in which we propose to target economically important traits like drought tolerance and virus resistance in grapevine.
Methods
Binary vector backbones for genome editing were acquired from Addgene. The targets for the selected LBCY gene were designed using the CRISPR-P and RGEN software. Constructs were assembled using a Gibson assembly strategy, and validated by Sanger sequencing before being transformed into Agrobacterium for plant transformation. Genetic transformation of N. benthamiana leaf disks and grapevine embryogenic callus was done according to routine protocols established at SAGWRI. N. benthamiana and grapevine plantlets were phenotypically (visually), genetically (PCR, RT-qPCR and sequencing) and biochemically (UPLC) analysed, according to established protocols.
Key Results
All molecular tools required for the editing of the proposed genes in N. benthamiana and grapevine were designed and constructed. These were used to generate a population of edited N. benthamiana plants were comprehensively characterised and demonstrated successful editing of the target gene. Both mono- and bi-allelic editing were observed. In spite of diminished regeneration ability, transformation with the editing constructs yielded 43 Chardonnay lines. However, molecular analysis performed on these showed that these were not transformed nor were they edited. The same construct was also transformed to grapevine in Italy, also leading to no edited plants, indicating that editing the specific gene could be lethal in grapevine. Another construct, targeting a different gene (VvPDS) was however successful, leading to the characteristic bleached phenotype, confirming that successful editing was achieved in this case. Transformation experiments with both Cas13a-LBCY vectors yielded working populations of N. benthamiana plants for both target sites. Preliminary RT-qPCR results on these lines showed reduction in the expression of LBCY in some samples but not consistently among all lines.
Key Conclusion of Discussion
Transformation experiments with a Cas9-based vector in N. benthamiana and comprehensive phenotypic, genetic and biochemical analysis confirmed the successful editing of these plants. Similar experiments with grapevine yielded regenerated Chardonnay plants, but genetic analysis on DNA from these plants showed failure of the transformation and thus also no mutations were introduced. Successful editing was obtained in a repeat experiment targeting a different gene that was conducted in Italy. Cas13a-based editing in N. benthamiana showed a reduction in expression of the target gene, but results were inconsistent and should be repeated with larger sample numbers.
Take Home message for Industry
In this project, we have demonstrated an ability to design and assemble constructs for the editing of N. benthamiana and grapevine, and in a very short time re-established a grapevine transformation and regeneration capability. This opens up possibilities for editing of grapevines to address biotic and abiotic stress issues of economic importance, such as virus resistance and drought.