How does gene editing work?
Gene editing is the insertion or deletion of sections of DNA. Some of these DNA segments represent genes that encode the instructions for making various cellular components. Other DNA segments control whether those instructions are carried out.
The technology for gene editing isn’t new. Scientists already figured out how to edit genomes back in 1973. But it was the 2005 discovery of CRISPR-Cas9 that revolutionised the field by making gene editing accessible to many more laboratories.
CRISPR-Cas9 is a naturally occurring part of the immune system of microbes. They use it to detect and destroy viruses (bacteria get viruses just like everyone else). The scientists who recognised the potential of CRISPR-Cas9 and developed it into a gene-editing system received the 2020 Nobel Prize in Chemistry.
“When you target a sequence, CRISPR-Cas9 cuts the double-stranded DNA, and then the repair system of the cell puts the strands back together,” explains Campa. “This causes a random modification of the sequence.”
That modification can inactivate (knock out) a gene or alter the extent to which the gene is expressed. Scientists control where CRISPR-Cas9 cuts DNA by designing guides that match the target DNA sequence. In the SATI-funded project, Campa focused on a gene called VvMyb60.
“Based on the literature, VvMyb60-like genes are linked to the regulation of stomatal opening and closing in other plants,” says Campa. “In those studies, the stomata are more closed, and there’s less transpiration when this gene is non-functional. And then the plants are more drought-tolerant because they lose less water.”
Although these studies were done in other species, the expression of VvMyb60 in grapevines is also linked to stomata, suggesting it would be a good target for gene editing to improve drought resistance.
Dialling down expression
The protracted process of creating genetically edited plants starts with tissue culture. Researchers culture grapevine anthers on artificial media to generate undifferentiated callus cells from the filaments. Undifferentiated cells can be manipulated with plant hormones to form shoots and roots, eventually giving rise to new little vines.
“You need to start from the callus because that’s the material you can edit,” says Campa. “But that callus takes a year to produce. When SATI approved this project, we didn’t have table-grape callus, so we started with Chardonnay callus.”
Since then, Campa’s laboratory has generated callus from table-grape cultivars, including Thompson Seedless and the 110 Richter rootstock.
“First, we targeted the VvMyb60 gene,” says Campa. “We transformed the callus cells and cultured them on different media, but we didn’t obtain any embryos.” Although it’s always tricky to know exactly why an edited cell isn’t viable, the most likely reason is that the VvMyb60 gene plays a crucial role during development.
Undaunted, Campa went back to the literature, identifying a second potential target: the promoter of the VvMyb60 gene. Promoters are like dials that increase or decrease gene expression. Other researchers have reported that inactivating parts of the VvMyb60 promoter, called DOF domains, reduced VvMyb60 activity.
A new round of editing aimed at two of the four DOF domains yielded viable plantlets. Next, Campa sequenced the plantlets’ genomes to verify that the genomes had been modified. She then checked VvMyb60 expression to confirm that it was reduced.
“Only one of the edited plant lines had a significant reduction in gene expression,” she says. “When we looked at the sequences, we saw that in some of the plant lines, the DOF domains didn’t change because the DNA was repaired. But in one line, the DOF4 domain was deleted.”
After successfully reducing VvMyb60 expression, Campa began evaluating the physiology of her edited plants. “We didn’t see what we were expecting,” she says.
Into the greenhouse
Using specialised software and a microscope, Campa compared the numbers and sizes of leaf stomata in edited and standard plants. To her surprise, the plants with the DOF4 deletion had a significantly higher stomatal density, and their stomata appeared larger and more open than those of the control plants.
However, these assessments were done on plants grown in the laboratory. To investigate plant performance, she conducted greenhouse trials comparing edited and control vines. Plants were taken from a well-watered to a severely drought-stressed state and then rewatered until they recovered.
“We saw that the line with the DOF4 deletion had a higher water-use efficiency and lost less water during drought,” says Campa. “Its photosynthesis was not affected by the editing.” She stresses that these are preliminary results and should be confirmed with more stomatal analysis and, ideally, more edited plant lines. “Unfortunately, these are projects that take a long time,” she says. “By the time you have an edited plant, three years have passed, and then you still need to start the full characterisation in the greenhouse.”
Nonetheless, this project demonstrates that it’s possible to apply gene editing to modify water-use efficiency and drought tolerance in grapevines. This is remarkable, considering that drought tolerance is a complex trait influenced by many genes. As Campa points out, traits influenced by single genes are far easier targets.
“For example, polyphenol oxidase, the enzyme that causes browning, has been edited in apples, avocados, and bananas. Why not do it in grapevines?” she asks. While based in Italy, she was involved in the research around Arctic apples, a series of non-browning cultivars grown commercially in the US.
Although Campa might find a three-year plant-production cycle painfully slow, genome editing still vastly outpaces conventional breeding. Given all its advantages, are we heading for a future in which genetic editing is the norm?
“There are still a lot of concerns about it,” remarks Campa. “But if you look at what’s happening internationally, genetically edited table grapes are coming.”
