Harvest time for CRISPR-Cas?

Abstract

The revolutionary gene editing technology CRISPR-Cas is on the verge of widespread application in agriculture, promising easy access to dramatic crop improvements. Companies now licensing the technology face the challenge of regaining the public trust that was lost in the GM debate. Additional insecurity comes from the ongoing patent dispute between two renowned US institutions. Michael Gross reports. The revolutionary gene editing technology CRISPR-Cas is on the verge of widespread application in agriculture, promising easy access to dramatic crop improvements. Companies now licensing the technology face the challenge of regaining the public trust that was lost in the GM debate. Additional insecurity comes from the ongoing patent dispute between two renowned US institutions. Michael Gross reports. Only four years ago, Jennifer Doudna and Emmanuelle Charpentier reported that the endonuclease Cas9, part of the bacterial immune system CRISPR-Cas, can be directed to edit specific sites simply by coupling it with a guide RNA. Soon after, other research teams, including those of Feng Zhang at MIT and George Church at Harvard, showed that this tool works in eukaryotic cells including human ones. The technique is revolutionary in its elegance, ease of use, and absence of side effects. The edits are typically indistinguishable from naturally occurring mutations. The immediate debate about the new tool focused on the application potential in human medicine and in particular in the human germline. The genomes of pre-implantation embryos could in principle be edited, raising the spectre of genetically engineered humans (Curr. Biol. (2015) 25, R439–R442). This issue is still unresolved but has recently been overshadowed by other applications of the method that are likely to surface in practice much sooner than any medical use might. Agricultural biotech companies have identified the tool as their new hope, enabling them to improve the genes of livestock and crops without the backlash produced by GM. Some gene editing has already addressed common problems in livestock farming. For instance, horns are mostly considered a nuisance and danger in dairy cattle, leading many breeders to remove them with rather brutal procedures. Naturally hornless breeds exist and are used for beef production, but are not suitable as dairy producers. With gene editing, the genetic disposition to grow horns can be readily removed from the genome of a dairy cattle breed, solving the problem once and for all, as researchers at the start-up company Recombinetics in St. Paul, Minnesota, USA, have demonstrated. The first hornless cattle produced by gene editing were born in 2015. Improvements of edible parts of farmyard animals are also under investigation. For instance, the teams of Huping Jiao and Daxin Pang at Jilin University, Changchun, China have bred pigs with increased muscle mass by inactivating the gene for the regulatory protein myostatin (Sci. Rep. (2015) 5, 16623). However, when genetic alterations affect the texture of the steaks, the fear of Frankenfoods is likely to raise its head again, along with concerns about animal welfare and about possible technology transfer from animal to human embryos. For these reasons, genetically edited meat products may face more of a struggle in terms of acceptance. In the UK, the Nuffield Council on Bioethics released a preliminary report on genome editing, concluding that the ethical implications of its use either in humans or in livestock require further scrutiny (https://goo.gl/Kxi97c). The situation looks generally more promising in plant breeding where small edits in crop genomes could lead to dramatic improvements in efficiency, disease resistance, and nutritional value of crops — the kind of improvements that global food production needs in order to keep up with the growth of world population and maintain food security in the second half of this century. Among the first gene-edited food products to reach consumers might be mushrooms. The group of Yinong Yang at Pennsylvania State University, USA, has developed a genetic procedure to stop common food mushrooms such as Agaricus bisporus — which is known under various names, such as button mushroom in its immature form and as Portobello in its mature stage — from turning brown on contact. The edit involves disabling one of six genes coding for the enzyme polyphenol oxidase. This could be an interesting test case for society and consumer response, but it is not going to have much effect on global food security. The big issues there are the world’s staple crops including rice and wheat, and the risks they are facing from disease and climate change (Curr. Biol. (2013) 23, R1–R4). Thus, researchers have harnessed the CRISPR-Cas system to do in plants what it evolved to do in bacteria, namely fighting viral infection. The group of Magdy Mahfouz at the King Abdullah University of Science and Technology at Thuwal, Saudi Arabia, has demonstrated this approach by immunising tobacco plants against tomato yellow leaf curl virus (TYLCV), a DNA virus from the family of geminiviruses, which pose a threat to food security especially in developing countries (Genome Biol. (2015) 16, 238). The groups of Jin-Long Qiu and Caixia Gao at the Chinese Academy of Sciences, Beijing, used a combination of two DNA editing techniques, TALEN (transcription activator-like effector nuclease) and CRISPR-Cas to make wheat resistant against powdery mildew, a widespread problem caused by a fungal infection (Nat. Biotechnol. (2014) 32, 947–951). Several Chinese laboratories are also actively developing tools for gene editing in rice, as preliminary publications and protocols document. Specifically, a recent report has broadened the applicability of CRISPR-Cas in rice by overcoming the requirement for an NGG motif near the target site (Mol. Plant (2016) 9, 943–945). Researchers at DuPont Pioneer, a genetically modified organism (GMO) producer based at Johnston, Iowa, USA, have used gene editing to improve the resilience of maize and soybean plants against environmental stress. The company Cibus at San Diego, California, has developed a rapeseed variant resistant to a specific weedkiller — an approach reminiscent of the much-criticised Monsanto strategy of “RoundUp-Ready” GM crops enabling farmers to use the herbicide RoundUp to kill off all other vegetation. With such echoes of the past, will the new technology fare better than the previous version? As more and more crop variants and livestock breeds are created by CRISPR-Cas gene editing, the big question is how regulators and consumers around the world will respond to these manipulations. In an important precedent, the US Department of Agriculture (USDA) has told Yinong Yang in April 2016 that his anti-browning mushrooms don’t require state regulation in the USA. This was the first such decision from the USDA for a variant created by CRISPR-Cas, but it follows the logic of several similar exemptions for work using the older technologies, TALEN and zinc finger nucleases. Essentially, the authorities believe that the controls introduced for GMOs in the 20th century are only necessary when foreign DNA is introduced into the manipulated organism, which was inevitably the case with the old GMO biotechnology. With the new technologies, changes can be as subtle as an exchange or removal of a single base pair. This means that the genetic alterations inherited by descendants of the targeted individual are indistinguishable from naturally occurring point mutations. Moreover, the precisely targeted nature of the change means that harmful side effects are less likely than in conventional methods used to speed up mutation rates and create a wider variety for selective breeding. In this context, mutagenic chemicals and radiation have been used, making the new technique look not only more elegant and efficient, but also green and sustainable. The European Union has yet to regulate on CRISPR-Cas products, but it appears plausible that it, too, may draw the line at the introduction of foreign DNA — if only because small edits are impossible to police as they look just like natural mutations. Whether consumers, especially in Europe, will be equally understanding remains an open question, especially considering the fierce opposition against 20th-century style GMOs in some European countries. For producers it will be crucial to communicate to consumers that the new products will be more natural than many things they bought before, and can in some cases even support more sustainable farming and reduce cruelty to animals. Another advantage could be in the fact that the new technology is so easy to use that any small start-up company can try its luck in the new field, and poorer countries could also develop their own biotechnology approaches. This more democratic approach to agro-biotech could conceivably gain back some of the trust that giant companies like Monsanto and DuPont lost in the GM debate, when they were accused of trying to make farmers dependent on their seed monopoly. In this context, it may not be entirely good news for the CRISPR technology that Monsanto has acquired a non-exclusive license for the use of the methods patented by the Broad Institute. Seeing that the invention of targeting Cas9 at DNA unrelated to its original function is generally credited to Doudna and Charpentier based on their seminal in vitro experiments (Science (2012) 337, 816–821), it comes as a surprise that most of the patents in the field have been granted to the Broad Institute, based on Zhang’s work with eukaryotic cells published only half a year later. While the University of California first applied for patents on the technology, the Broad Institute applied for a fast-track treatment and got their patents granted in April 2014, against which the University of California filed an appeal a year later. What the patent office will have to clarify is whether the later work with cells is a simple and logical application of the earlier in vitro work, or whether it constitutes an independent invention. At the time of going to press, the case is still wide open. A number of biotech start-ups that have built their business plans on licenses obtained from the Broad may have to go back to the drawing board if the institute loses its patents. Apart from the business implications, the patent dispute may also have changed the course of Nobel Prize history. Doudna and Charpentier would have been strong candidates for the chemistry prize in 2015 or 2016, but Stockholm will now likely await a definitive decision on the patents before the inventors can reap their rewards.