Winetech Technical Yearbook 2022

APPLICATIONS OF CRISPR The range of applications is wide: in cluding biological research, breeding and development of agricultural crops and an imals, and human health. Before focusing on the agricultural/viticulture applica tions, it is worth it to mention an example of medical use of the technology. CRISPR-Cas9 was used to correct mutations associated with the human genetic disease, β-thalassemia. First, induced pluripotent stem cells from the β-thalassemia patients were created. Subsequently, CRISPR-Cas9 was used in these cells to correct the mutations in the non-functional human hemoglobin beta gene, resulting in cells with restored expression of the human hemoglobin beta gene, which can be used for gene therapy. Regarding agriculture, it is crucial to mention that evidence suggests that through the potential contributions to increase yield, enhance nutrition and greater environmental sustainability, genome editing can help attain the top three Sustainable Development Goals (SDGs) identified by the United Nations. For example, genome editing can contribute to increase sustainability through improvement of water- and nitrogen-use efficiency of crops, reduction in environmental footprint in agricultural production, and enabling the production of more food using less or the same amount of resources as conventional crops and livestock. APPLICATIONS IN GRAPEVINE Uptake of CRISPR technology in the grapevine research community industry has been slow and cautious, probably because of the notorious recalcitrance of grapevine, being a perennial woody fruit crop, to any type of genetic improvement. The uncertain and contradictory policies regarding the regulation of genome edited crops in different parts of the world certainly also contributes to this slow progress with applications in grapevine. Ironically, the precision and relatively low cost of the technology may be particularly useful in the wine industry, where a huge value is attributed to existing Vitis vinifera varieties. Unlike in the case of traditional breeding, where genetic backgrounds are randomly mixed, the minimalistic nature of the genetic modifications (most of the time the removal or insertion of a single genetic letter) introduced by CRISPR technology, allows for desired changes to be made, while retaining cultivar characteristics. CRISPR/Cas9-edited grapevine plants have been generated in a

few cases. In 2016, CRISPR-induced point mutations in ‘Chardonnay’ embryogenic cell masses led to the regeneration of plants with an altered production of tartaric acid and vitamin C. Around the same time, attempts to create non transgenic edited grapevines by directly delivering purified Cas9 and gRNAs into ‘Chardonnay’ cells, generated edited cells, but could not recover viable plants. In parallel, the technology was used to create edited grapevine plants using two different delivery systems, one GMO and one DNA free. In another application, ‘Neo Muscat’ plants displaying the characteristic albino phenotype in leaves were obtained after somatic embryos were transformed with a CRISPR/Cas9 editing construct targeting the phytoene desaturase gene. A number of applications also targeted major diseases of grapevine e.g., transgenic ‘Thompson Seedless’ plants, in which the WRKY52 transcription factor gene was mutated using CRISPR/Cas9, were recently produced for increased resistance to Botrytis cinerea. Likewise, the technology was used to edit both downy mildew and powdery mildew susceptibility genes in the powdery mildew-susceptible cultivar ‘Thompson Seedless’ and in different grapevine clones. In other food crops, CRISPR/Cas technology have been used to achieve impressive results, mostly to address biotic and abiotic stress conditions. Examples include drought and salinity stress in many grain and vegetable crops, and numerous fungal, bacterial and viral diseases in crops like maize, wheat, rice, potato, tomato, soybean, cabbage and cucumber. Recently, an unexpected feature of the alternative Cas12, Cas13 and Cas14 nucleases, that of non-specific activity upon recognition and digestion of the original target, has been exploited to design diagnostic systems far more accurate and sensitive than any existing methods. WORLD VIEWS OF THE TECHNOLOGY Policy regulations for the CRISPR-Cas9 system vary around the globe. As far as agriculture is concerned, countries are regulating the production of edited crops in different ways. More conservative approaches, like in Europe, consider genome-edited crops as GMOs, simply because of the processes used for producing them, and therefore they are assessed by the cumbersome GMO regulatory processes. In other countries, like the USA, if the crop was produced by introducing a genetic mutation, without

the introduction of foreign DNA, these are not subjected to any regulation. South Africa is following the lead of Europe and edited crops will be regulated like GMOs. It is important to avoid the same mistakes made with GM crops, and to allow genome-edited crops to be more accessible to small/medium enterprises, that can’t afford the burden of unnecessary and onerous regulatory processes. In a recent comment paper in the influential Nature Genetics journal, scientists recommended that policies are adopted to support the use of genome editing technologies and genome-edited crops to help improve the livelihoods of smallholder farmers. The need for decisions on the regulatory, trade and intellectual property frameworks are urgently needed to ensure that the best that science can offer, contributes to equity and is not available only to the privileged or wealthy. ABSTRACT CRISPR/Cas-based genome editing is a technology that can modify the genetic code of any organism in a precise way, and in a manner that does not require the insertion of foreign DNA into the genome of the target organism. Since its invention almost a decade ago, CRISPR/ Cas technology has found a multitude of applications in medicine and other industries. In agriculture, the technology is seen as an essential part of any approach to address food sustainability towards the second half of this century. Desirable traits like tolerance to adverse abiotic stresses such as drought and salinity, which are associated with a changing climate, as well as resistance to pests and diseases that threaten food production, can realistically be achieved using CRISPR/Cas technology, and in a fraction of the time compared to conventional breeding approaches. Several impressive successes have been recorded in many crop plants over the last few years, including a few in grapevine. REFERENCES https://www.wineland.co.za/the-crispr revolution-and-grapevine/

For more information, contact Manuela Campa at mcampa@sun.ac.za.

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WINETECH TECHNICAL YEARBOOK 2022