- Biotechnology has a new cause célèbre that supersedes GM technology in much the same way that smartphones overtook cell phones
- Called gene editing, the new method is more subtle and precise than anything that precedes it
- The new method is being applied to crop science, including a GRDC project in Australia that is allowing a backlog of genetic insights to now find applications in pre-breeding research
Dr Brian Jones with GRDC-funded technical officer Helen Le examining a Brassica rapa line in the University of Sydney laboratory that has the highly promising new technique, gene editing, working routinely.
PHOTO: University of Sydney
Even though the GM debate drags on, unresolved, it is becoming less relevant in the face of new science – gene editing – which could offer human medicine and crop and livestock science unprecedented advances without the lingering DNA baggage that fuelled opposition to genetic engineering
There is a buzz in scientific circles about gene editing, a suite of new techniques that can precisely target changes to the DNA sequence in human, plant or animal genomes.
The technique works by guiding the cell’s own DNA repair mechanism into making a modification to the genome. Among the changes made possible by gene editing is the ability to repair, silence or rescript any segment of DNA, including genes.
Compared with GM technology, gene editing could amount to a step change in capability, efficiency and precision that leaves normally level-headed scientists unusually excited. Experts are describing recent breakthroughs in gene editing as “transformative”, “disruptive” (rendering previous technologies obsolete) and, according to the journal Nature Plants, it is creating the “dawn of a new paradigm in plant breeding”.
Early achievements have been genuinely impressive. Included is a human trial of HIV patients in 2014 that saw half of the participants able to stop taking anti-viral drugs since gene editing increased the resistance of immune cells to the virus by inactivating a human gene used by the virus to infect these cells. In leukaemia trials this year in the UK, 80 per cent of participants experienced a loss of symptoms and half achieved remission.
And these trials used a far less nimble version of the technique that is available now, so rapid is the technology’s development.
What remains true of all versions of the technique is that, unlike GM technology, it leaves no trace of the DNA constructs that were used to guide the genome modifications.
As such, gene editing erases the fractious distinction between GMOs and conventional organisms. Regulators in the US have already decreed that GM regulatory regimes (and their associated costs) do not apply to gene-edited organisms. Australia and the EU are now reviewing their own regulations.
In the interim, Australian regulators are allowing researchers to use the technology under laboratory conditions and the GRDC has funded Dr Brian Jones at the University of Sydney to trial gene editing in canola.
He is using the technique to modify a single canola (Brassica napus) gene so that it mimics a mutation discovered in a related species, Arabidopsis thaliana. In Arabidopsis, the mutation is associated with improved yield and water use efficiency. (Arabidopsis is a small flowering plant commonly used as a model for studying plant biology.)
Irrespective of whether Dr Jones’s gene-edited canola is ultimately subject to the same restrictions as GMOs, scientists say the important fact is that gene editing works, and in ways that open a floodgate of innovation possibilities.
“These extraordinary techniques have finally given us the tools we need to apply the accumulated insights into how genetic diversity affects crop performance,” Dr Jones says. “Field and laboratory studies on breeding material, landraces, wild relatives and model plant systems have produced a mountain of detailed insights about important variations in single genes, or gene networks, and how their application could benefit agriculture.”
The previous bottleneck in the system was an inability to efficiently transfer that knowledge into commercial cultivars cheaply, quickly and effectively. “No previous technology had the kind of scope that gene-editing techniques have given us. As a molecular geneticist, this is the capability I have been waiting for.”
How to edit a plant gene
Both gene editing and GM technology start by introducing new DNA into living cells. In GMOs, the foreign DNA incorporates at random sites in the cell’s genome, introducing all the DNA necessary for the sought-after gene manipulation.
With gene editing, the introduced DNA is needed only temporarily. It is introduced to select the specific region in the genome to be modified and to introduce a break in the DNA at that site. Once the cut is made, the introduced DNA constructs are no longer needed and can be dispensed with.
Gene editing for beginners: A DNA-cutting protein (the scissors) allows researchers to 'edit' genes (and their traits) as the genome self-repairs
The cut brings the cell’s own DNA repair mechanisms into play to make repairs. Depending on which of the cell’s internal repair mechanisms are used, a targeted gene can either be silenced, rescripted to add new properties, or modified so that extra genetic information is added.
For instance, silencing a gene – such as a disease susceptibility gene in a plant genome – can be achieved by cutting the DNA within the gene’s ‘coding’ region (the region in which the protein causing the disease susceptibility exists).
“You allow the cell to repair the DNA so that it simply joins the two cut ends back together,” Dr Jones says.
“That repair leads to a gene that no longer specifies a functional protein, in this case, a protein responsible for making the plant susceptible to a particular disease.”
It is the ability to finesse the natural repair processes that results in different outcomes at the targeted DNA site.
Currently, three different ways have been developed to guide a DNA-cutting protein to the targeted region of a genome (Table 1).
Two are based on reprogramming DNA-binding proteins so that they will recognise the specific site of interest. This is the approach taken with the zinc finger nuclease (ZFN) and TALEN® methods.
Neither of these methods, however, excites Dr Jones as much as the nimbler, subtler, faster and cheaper third method: CRISPR/Cas9.
Rather than using complex DNA-binding proteins to recognise a gene, CRISPR/Cas9 uses a simple RNA guide. RNA is the molecule produced once any gene, including a transgene, is expressed within a cell. The guide RNA is designed to introduce two functional elements. One element corresponds to the targeted site in the genome, binding to it (somewhat in the manner of a gene marker). The other provides a binding site for the protein that cuts the genomic DNA, in this case, the bacterial Cas9 protein is used.
“The protein-driven approaches were real breakthroughs, but they are expensive, time consuming and laborious,” Dr Jones says.
“But the cost has fallen dramatically with the advent of CRISPR-Cas9.
“The basic machinery for the protein-based techniques would typically cost $5000 to $10,000. The CRISPR/Cas9 guide RNA can be synthesised for about $40.
“This means that a number of sites can be readily targeted, in the one gene or throughout the genome. This is why the potential of CRISPR/Cas9 really is exciting.”
Table 1 The three faces of gene editing.
| Zinc finger nucleases
(Transcription activator-like effector nuclease)
(Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated protein-9 nuclease)
|What is it?
||A protein consisting of a DNA-cutting enzyme and a DNA-grabbing region that can be tailored to recognise different genes.
||A protein consisting of a DNA-cutting enzyme and a DNA-grabbing region that can be programmed to recognise different genes but is easier to design than zinc finger nucleases.
||A DNA-cutting protein guided by an RNA molecule to single out the gene of interest.
|Pros and cons
||It was the first programmable genome-editing tool but it relies on proteins that can be difficult to engineer for new gene targets. Potentially dangerous off-target cuts are also possible.
||Although simpler and cheaper than zinc finger nucleases, TALEN proteins can still be difficult to produce. Off-target cuts are a problem.
||This technique is affordable and easy to use, and it works for high throughput, multi-gene experiments. Different Cas9 proteins can reduce the potential off-target cuts.
SOURCE: Susan Young, MIT Technology Review
The first commercial plant-breeding application of CRISPR/Cas9 resulted in a canola variety that was released in the US in April 2015. The modification, however, involved an all-too-familiar trait: herbicide resistance.
Dr Jones at the University of Sydney has something more ambitious brewing in his state-of-the-art growth chambers, although he too is targeting canola.
His project builds on the discovery of a single mutated Arabidopsis gene – called AtA18-1 – that leads to enhanced photosynthetic and water use efficiency, along with an increase in total seed yield of about 37 per cent per plant.
“Here we have a case of variation at a single gene that leads to some extraordinary changes, including improvements in drought tolerance, photosynthetic capacity and seed oil content,” Dr Jones says.
“Our work suggests that the gene is a central regulator of growth and development and a potentially valuable target for breeding work. What gene editing allows us to do is directly target the analogous gene in canola and rescript it so that it mimics the AtA18-1 variant in Arabidopsis.”
By rescripting it, the canola gene acquires the same features that result in greater photosynthetic and water use efficiencies that were observed originally in Arabidopsis.
Dr Jones is using CRISPR/Cas9, a method he has up and running routinely in his laboratory. But how he is using it reflects a dedication to creating knowledge about genetic diversity and accelerating its applications to crop-breeding programs.
The AtA18-1 gene project illustrates this. The gene variant was discovered when Dr Jones was based in Sweden as part of a project investigating tree growth. It was one of six gene variants that affected plant growth which he brought to Australia to study when he took up his current position in Sydney.
He has now introduced AtA18-1-like changes to the analogous gene in several oilseed species using a conventional method, and gene editing. He reports the experience of using gene editing as “liberating”.
“Single gene variations that bring benefits to growers have been identified in all our major crop species,” he says. “CRISPR/Cas9 allows us to use that knowledge directly.
“Gene editing is also applicable to more complex ‘multigenic traits’, in which many genes contribute to a beneficial trait, such as WUE. That’s important. The work we’re doing must have beneficial impacts for growers and food security. Efficiency matters a great deal.”
It has now been shown that CRISPR/Cas9 constructs can be introduced into cells ‘transiently’ so that they never insert into the genome while they carry out the genome modification, Dr Jones says. “That means they can be subsequently eliminated without the need for back-crossing and segregation steps.”
Multiplexing tools have also been developed to target multiple sites in a genome for consecutive editing in one hit. This will allow agronomically important ‘multigenic’ traits to be targeted efficiently.
Galvanising the innovation is the willingness of scientists to share CRISPR/Cas9 methods and materials without intellectual property restrictions. Credit for this collaborative agreement belongs to Dr Jennifer Doudna, the biochemist at the University of California, Berkeley, who invented the CRISPR/Cas9 system.
Dr Doudna made the breakthrough while studying how bacteria recognise and disable viral invaders using a protein (Cas9) that she compared to a “genetic scalpel” since it slices up viral DNA.
In 2012, she reported that the action of the Cas9 scalpel protein was programmable, allowing it to be targeted to any gene in any species using a job-specific designed CRISPR guide RNA.
“Direct, precise editing of the genome has been the holy grail of molecular genetics,” Dr Jones says. “The power of CRISPR/Cas9 means that we can expect to see a rapid uptake of the technology and a growing list of innovative uses. It is easy to get excited about the possibilities for breeding. Hopefully, growers will be reaping the benefits before too long.”
Dr Brian Jones, Department of Plant and Food Sciences, University of Sydney,
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