This review will focus on one promising innovation in breeding technology, namely genome editing.

Agriculture is critical in addressing some of the world’s major challenges: food shortage, nutrition, health and sustainability. Most of the advances in agriculture in the last century can be attributed to either mechanisation (industrial revolution) or improved management and plant breeding and selection (green revolution). Further improvement in productivity and sustainability will, however, require continued advances and innovation.

 

What is gene editing?

Genome editing is a catchall phrase to describe the technologies that are available to alter an organism’s DNA. The most promising of these technologies is CRISPR, which is an acronym for ‘Clustered Regularly Interspaced Short Palindromic Repeats’ and occurs in nature as a bacterial ‘immune system’.

The technology is relatively simple and versatile, hence its success in revolutionising science, from medicine to agriculture. A decade ago, Jennifer Doudna and Emmanuelle Charpentier unveiled the potential of this technology and since then the progress and applications increased exponentially.

This discovery is having such an impact on our lives that the two scientists were awarded the Nobel Prize for Chemistry in 2020.

Much like antibodies in our own immune system, the CRISPR system enables the bacteria to identify an infecting virus and target specific sequences for destruction using specialised proteins. These proteins are called CRISPR-associated proteins (Cas).

 

How does gene editing work?

Doudna and Charpentier demonstrated that the Cas protein can easily be re-programmed to find and bind to almost any desired target sequence, simply by attaching a piece of RNA (guide RNA) to steer it in its search.

When the Cas protein and a guide RNA are delivered into a cell, they form an editing complex that moves along the strands of DNA until it finds and binds to a 20-DNA-letter long sequence that matches part of the guide RNA sequence (Figure 1).

This is an impressive feat, considering that the DNA packed into each human cell has six billion letters and is two meters long. Once the target sequence has been recognised, the Cas protein cuts the DNA at the target site.

The cut gets repaired by one of two natural repair mechanisms, the most common of which is error-prone, resulting in a knock-out mutation that usually disable the target gene.

Another repair mechanism relies on homology repair and can be used to introduce foreign DNA, creating a knock-in mutation. So, for instance, scientists could program the Cas protein to snip out a gene that causes Huntington’s disease in humans and insert a “good” gene to replace it.

Numerous types of CRISPR/Cas systems have been discovered – the one we described so far is the most widely used and well known: CRISPR/Cas9. Several more Cas proteins have been characterised that have slightly different mechanisms and target requirements. For example, Cas13, can cut RNA instead of DNA.

 

FIGURE 1. CRISPR/Cas system and repair mechanism for gene editing by targeting specific DNA sequences.

 

Advantages and disadvantages of the technology

Various techniques to knock out genes have been around for years. What makes CRISPR so revolutionary is that it’s so precise: the Cas protein mostly goes wherever it is directed. And, as mentioned before, it’s incredibly versatile, cheap and easy. Also very promising, this technique has worked on every organism it’s been tried on: animals, plants and bacteria.

It provides the possibility to cure genetic diseases and to produce plants that can resist pathogens and adverse climate conditions. CRISPR can be used for DNA-free gene editing without the use of foreign DNA. A DNA-free gene editing system can be a good choice to avoid the possibility of undesirable genetic alterations due to the foreign DNA integrating randomly.

In spite of being such a recent discovery, CRISPR technology has already achieved incredible results. However, each CRISPR system needs to be optimised to be accurate and reliable, especially in eliminating the so called ‘off-targets’ edits. Indeed, it has been observed that sometimes a sequence not exactly the same as the one intended, can be recognised and cut by the CRISPR/Cas.

Like all powerful technologies, it is important that safety and ethical aspects are considered. Scientists must consider that there are potential hazards of using this technology in an irresponsible way.

 

Applications of CRISPR

The range of applications is wide: including biological research, breeding and development of agricultural crops and animals, and human health. Before focusing on the agricultural/viticulture applications, 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.

 

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

 

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