News - September 22, 2016

Cutting and pasting genes

Rob Ramaker

The new technology Crispr-Cas9 makes it easier than ever to edit genetic material with great precision. The development of this gene editing technology is moving so fast that legislation and ethics are having trouble keeping up. Is this gene manipulation or accelerated classic breeding?

Illustrations JeRoen Murré

‘Today we are learning the language in which God created life,’ said the then American president Bill Clinton in June 2000. He was receiving the results of the Human Genome Project, in which all the genetic material of a human being – all the genetic letters A, C, G and T – was decoded for the first time.

Now, 16 years later, scientists can read this ‘gene language’ increasingly fast, easily and cheaply. And in the past few years they have even managed to edit this language with increasing success.  A new technique called Crispr-Cas9 has made it simpler than ever to tinker with the genetic material – the DNA – of plants, animals and humans. This makes it possible, for instance, to prevent hereditary diseases or to give plants new characteristics.


The story of Crispr starts in a bacterium. Although themselves pathogens, these micro-organisms can also fall sick if they are infected by malignant viruses. Some bacteria, however, have developed a unique defense mechanism against this. They store up little ‘barcodes’ – Crisprs – of the pathogenic viruses they encounter. Proteins such as Cas9 – there are other variants too – then scan the cell for these barcodes. When a pathogen is detected, Cas9 mercilessly cuts out the genetic material of the virus. Enemy eliminated.

John van der Oost, personal professor of Microbiology at Wageningen University, is one of the pioneers of Crispr technology. His researchers showed how the system works in relation to the protein Cascade. Another variant – Cas9 – turned out to be easier to use as a biotechnological tool that Cascade. In the lab the protein uses a tailor-made ‘guide’ to find specific locations in the genetic material and make a cut there. This enables scientists to switch off genes, replace fragments of genetic material or correct genetic letters – a process they themselves call genome editing.

As soon as the potential of Crisprs became clear, teams all around the world applied themselves to the technique. In no time it was used on plants, zebra fish, mice, and eventually even on human embryos. The ‘revolutionary’ and ‘Nobel prize-worthy’ technology elicited unprecedented enthusiasm all around the world. At the end of 2015, the scientific journal Science declared Crispr-Cas9 ‘the breakthrough of the year.’ The technique is expected to have a massive impact on science and beyond it.

Long life tomatoes

Plant breeders are watching the rapid development of Crisprs with interest. ‘I have never seen anything like it,’ says Jan Schaart, a researcher at Wageningen Plant Research. Schaart already had some experience with earlier methods of genome editing, using specially developed proteins, for instance. These were extremely labour-intensive compared with Crispr-Cas9. ‘One of the strengths of Crispr-Cas is that it is so easy.’

This view is shared by Ruud de Maag, another researcher at Wageningen Plant Research. He has been using the method since 2015. De Maagd used Crispr-Cas9 to cut pieces out of genes to disable them – by far the most common application. ‘We got results very easily,’ he says. ‘At once, in fact.’ His ultimate goal was to improve the shelf life of tomatoes. The ideal is to create a variety that goes soft less quickly without slowing down other aspects of the ripening process, such as flavour development. To achieve this, De Maagd needs to find out which genes determine individual characteristics. The easiest way to do this is to switch off the genes and see what happens. His fellow plant scientist Schaart envisages other applications too, such as making plants less vulnerable to diseases. This is done by switching off genes that pathogens need in order to infect plants. It’s as though you remove an Achilles’ heel. But it can only be done if the plant can do without that gene. ‘More and more of these kinds of susceptibility genes are being discovered,’ says Schaart. ‘They create robust resistance.’  Schaart would like to modify oil crops too, so that they make a more useful oil mixture for human use.

One of the strengths of Crispr-Cas is that it is so easy


Although Crisr-Cas9 is simplifying his work, Schaart does not think it will entirely replace existing breeding methods. ‘We’ll go on needing classic breeding through cross-breeding and selection.’ Crispr-Cas9 will help with creating targeted new mutations – and thus variation. This is currently labour-intensive and random work. Plants and seeds are first ‘mutagenized’: blitzed with radiation or exposed to a chemical which causes hundreds of mutations. After that thousands – or even tens of thousands – of plants are screened for the right mutation. This is where Crispr-Cas9 provides a short cut, with far fewer unsolicited mutations.


Plant breeders would also like to replace (fragments of) genes in plants with more useful variants, or introduce new material with Crispr-Cas9. In practice this is still difficult, though. Plants only seem to build in newly introduced genetic material incidentally. But both Schaart and De Maag are optimistic that new innovations are going to make this possible.

Crispr-Cas9 is still a new method and its full potential has yet to be discovered. New or better applications appear every month in scientific journals. One possibility opened up by combining Cas9 with another enzyme is to change individual genetic letters. ‘My imagination can really run wild on the potential of that,’ says Schaart. What is more, new Crispr systems with new potential are still being discovered. One example is Cpfl, a protein described by John van der Oost and a group of American researchers. This protein resembles Cas9 but cuts out genetic material in a different way. A further possibility is to change Cas9 so that it does not switch off genes but either counteracts or actually stimulates their functioning.

GMO or not?

As the technology advances by leaps and bounds, legislation cannot keep pace. The European Union makes a strict distinction between plants produced by classic breeding and by genetic modification (mutagenesis using, for example, radiation, comes under the latter category but is exempted because it has been used safely for decades). This distinction is becoming harder and harder to make, concluded the Dutch Commission on Genetic Modification (Cogem) earlier this year in its Biotechnology Trend Analysis. This is due not only to Crispr-Cas9 but also to other innovative breeding techniques. The commission therefore proposes that it is time to ‘review the EU legislation to provide clarity for the public, consumers and industry.’

The current lack of clarity poses few problems to scientific research. Yet it can throw up limitations. For example, a project proposal by Plant Breeding was rejected after strong criticism by the evaluating committee, says René Smulders, business unit manager at Wageningen Plant Research. The committee felt the status of the Crispr-Cas9 technique was too uncertain. A bitter disappointment, says Smulders, because the call by Horizon2020 explicitly aimed at innovative breeding techniques. Schaart notices that clients in the business world are also worried about whether the applications under consideration will be subject to the legislation on genetically modified organisms (GMOs). A situation considered unworkable by most companies.


Brussels promised more clarity last December. The decision was then postponed until March 2016, only to be postponed again. Now some member states are going their own way. The Swedish Board of Agriculture, for instance, licensed the cultivation of cabbage in which a gene had been switched off by Crispr-Cas9, but no ‘foreign’ DNA had been introduced. An unprecedented move in Europe. The right decision, according to the researcher responsible, Stefan Jansson at the University of Umeå. As a statement he publicly consumed the product, in the form of Tagliatelle with ‘Crispry’ fried vegetables. Most researchers express themselves less theatrically but share the conviction that it is absurd to define a plant as a genetically modified organism when the mutations in question could also come about naturally. They would rather have Crispr-Cas9 any day than the crude mutagenesis brought about by chemicals or radiation. Schaart and many of his colleagues want to get rid of the process-driven assessment that looks at how a plant is produced. In its place they would like to see a product-driven assessment. If the mutations in question could have come about naturally, then they see no reason to subject the plant to the strict GMO legislation.

Scientists want the product to be assessed, not the process

Greenpeace took the opposite position in November last year. The organization claims that although gene editing is indeed more precise that earlier forms of genetic modification, ‘newly created organisms could display unexpected and unpredictable effects’. Products which contain GMOs should therefore be labelled and traceable. And they should be subject to strict legislation. In the eyes of Greenpeace, modification in the lab is categorically different to mutations which arise spontaneously, and even to mutations brought about through mutagenesis by chemicals or radiation.


Scientific discoveries have proceeded exceptionally fast in the case of Crisprs. Discussion of the ethical implications and legislative framework, on the other hand, has only just started. Plant breeders and scientists hope to avoid the intense polarization that arose around GMOs in Europe in the 1990s. Now that the general public is getting to know about the technique, it will be interesting to see how they react to it.

Back in 2000, President Clinton expressed concern about the power that came with knowledge of our genome. But optimism nevertheless prevailed – prematurely at that point perhaps, but with more and more basis in reality since. ‘The science of the genome,’ reckoned Clinton, ‘is going to have an enormous impact on our lives.’


Hornless cattle


American scientists demonstrated earlier this year that they had used gene editing to create a cow without horns. All they had to do was to introduce a single gene variant. That may sound like a miracle, but Martien Groenen, personal professor of Breeding and Genetics, is cautious. He fears the potential of gene editing for livestock breeding may be being exaggerated.

The reason is that in order to keep sufficient genetic variety and prevent inbreeding, breeders have to work with sizeable herds. If they want all their animals to get a new gene – for hornlessness, for instance – they either need to modify the genome in many embryos, which is expensive and far-reaching, or to select so stringently for the new gene that they lose other positive characteristics. This makes a new breed much less interesting.

So gene editing is not yet viable in livestock breeding, unless a new gene makes an animal massively more interesting to farmers, says Groenen. He does see a lot of potential in the scientific research.


Crispr pioneers in Wageningen

Jennifer Doudna, one of the pioneers of Crispr-Cas9, is coming to Wageningen on 30 September. She is in the Netherlands to receive the Heineken prize for biochemistry and biophysics. She will give a lecture together with Edze Westra, winner of the Heineken Young Scientists Award and an alumnus of Wageningen University. In the lecture in Orion the pair will describe their work and the ethical dilemmas it raises. Doudna was invited to Wageningen by Science Café Wageningen, Resource, the Microbiology chair group, Wageningen Young Academy and the KNAW. You can register for the lecture on the website of Wageningen Young Academy.