Genome editing technique: CRISPR-Cas9 and its role in agriculture

Potential applications and implications

CRISPR-Cas9 is a tool for very precisely engineering an organism’s genetic material (see the next section for a more detailed explanation). Thousands of research papers using CRISPR-Case9 to engineer different cells in many different organisms have been published since early 2013 (the CRISPR-Cas9 technology was first described in 2012). As well as its applicability as a fundamental biological research tool in laboratories (by allowing scientists to knock-out or subtly change genes of interest and observe the results, to uncover the underlying mechanisms of biological processes – which they currently do using conventional genetic engineering techniques), the CRISPR-Cas9 system has been hailed for its wide array of potential “real-world” applications. In particular, many are interested in its medical applications, for example in correcting disease-causing genetic mutations, engineering pigs to produce human organs to use for transplant, or less directly, for introducing deleterious mutations into malaria-carrying mosquito populations to reduce the population and control the spread of malaria. A clinical trial in China involving the injection of CRISPR-Cas9 edited cells into cancer patients was the first case of CRISPR-Cas9 being used directly in humans (Nov 2016).

Outside of the medical field, people are interested in the potential applications of CRISPR-Cas9 in solving some of the trickiest genetic engineering challenges, such as producing bacteria that can carry out all of the steps necessary to break down tough plant material (like cellulose) to produce biofuels. Increasingly, the potential applications of CRISPR-Cas9 to agricultural problems are coming under scrutiny, particularly in the context of crop improvements (increased stress tolerance, yield, nutrients, etc.), but also in relation to livestock engineering. For example, researchers are investigating the possibility of using CRISPR to engineer pigs that are immune to a particular haemorrhagic virus that devastates farms in Sub-Saharan Africa and Eastern Europe (see the Ensia article linked below for more on this), or to make chickens which lay hypoallergenic eggs.

It goes without saying that none of these proposed applications are without controversy. Aside from the direct implications of the applications of CRISPR-Cas9 technologies (i.e. for fundamental biological research, health and the environment), CRISPR also has potentially massive implications for the public debate and perception – and the regulatory and legislative structures – concerning genetic engineering as a whole. CRISPR-Cas9 raises questions about what general publics are willing to accept, whether in food, medicine or elsewhere, as well as questions about what defines a genetically modified organism, what level if any of genetic editing is permissible in human populations, and whether there are slippery slopes from correcting lethal genetic mutations all the way to eugenics or “designer babies”. Some of these issues are addressed by the resources listed below.

Brief scientific background

The CRISPR-Cas9 technology is derived from a bacterial defence mechanism against viruses. It allows bacteria to store a copy of a virus’ DNA in their own genome, which then allows for the quick detection and destruction of that virus’ DNA, should the bacteria encounter the virus again. This can be considered analogous to our own acquired immunity against diseases (only in the broadest sense of our bodies recognising a disease-causing agent quicker if you’ve encountered it before – the mechanism is very different). In the original natural bacterial CRISPR system, bits of virus DNA are stored in a part of the bacterial genome called the CRISPR region (which stands for “clustered regularly interspaced short palindromic repeats”). From this DNA, temporary copies called RNA are made continuously. RNA made from the CRISPR region acts as a guide (and is in fact called “guide RNA”) for enzymes called Cas enzymes. These enzymes ‘hold’ the guide RNA and ‘patrol’ the bacterium, testing any encountered DNA against the guide RNA template. If a match is found (i.e. if the virus with matching DNA is present), a specialized part of the Cas enzyme acts like a pair of molecular scissors and cuts the DNA of the virus (by breaking the chemical bonds holding together the main ‘backbone’ of the DNA molecule) so that it no longer works (much like diffusing a bomb). So, a short summary of the naturally occurring system: a copy of virus DNA is stored in the CRISPR region, from which guide RNAs are produced; these RNAs form a complex with a Cas enzyme, which uses it as a template to locate and destroy matching virus DNA.

The co-opted version of this system, which can be used by scientists to edit genes precisely, involves using synthetic guide RNAs (i.e. sections of RNA designed to match a specific section of a gene that you wish to edit) – instead of RNAs made from stored virus DNA – and a specific Cas enzyme called Cas9 (usually a slightly edited version of a naturally occurring Cas enzyme from the bacterium Streptococcus pyogenes, although others can be used) (see Fig. 1). As the DNA and RNA codes are universal across all known life, guide 

RNAs can be produced to match any gene sequence of interest, meaning that, in theory, the system can be applied to any organism of any species. This is part of the power of CRISPR-Cas9 and one of the reasons that it is causing such excitement among scientists (aside from its many potential exciting applications). The Cas9 enzyme cuts both strands of the DNA in the region of the genome specified by the guide RNA. During the subsequent DNA repair process, specific changes can be introduced to change the function of the gene in the desired way. This could entail large changes – like introducing new genetic material (such as a whole gene from another organism, which is a very precise method of genetic modification), or introducing DNA that disrupts the whole gene, “knocking out” its function – or more subtle changes, such as switching one or a few base-pairs in the gene to very slightly modify the function of the gene’s product (e.g. the protein made using the instructions encoded by the gene). Clearly, this combination of guide RNAs and specific enzyme activity provides an extremely precise and powerful tool for modifying genomes, with many many potential applications, as described above. 

Recommended resources

Mining through the swathes of scientific articles and alternative perspectives of any new technology can be a daunting task, so the FCRN has gathered here for you some of what we view as the most informative and useful resources on the topic of CRISPR-Cas9 technologies and their potential applications in agriculture, to enable our members to feel well-informed on this topic and able to form reasoned perspectives on its implications for food sustainability.

• **Highly recommended reading** For an excellent, balanced and thorough overview of CRISPR, its potential applications in agriculture and in particular its implications for the discourse on GMOs, read Maywa Montenegro’s January 2016 article in Ensia, here.
• Videos: For a comprehensible and to-the-point introduction to the scientific background of CRISPR-Cas9 technologies, we recommend this animated video by Alila Medical Media (run time: 5min30). The first four mins in particular provide a useful factual overview of the bacterial CRISPR system and how it can be used as a gene editing tool. However, it is important to note that this video is made by a US-based media company, and so the final minute and a half of the video discusses some applications which would not be legal in most countries outside of the US (for example, designer pet fish are discussed, which are legally sold in the US, but are illegal in most developed countries). For a longer and more detailed introduction to CRISPR-Cas9 technologies and their background, see this video by Kurzgesagt (run time: 16.03) – do bear in mind, however, that its slight hyping and overstating of the technique, its applications and of scientists’ intentions with it (both good and bad) should be taken with a healthy pinch of salt.
• This article by the Berkeley News reports on the expansion of the Innovative Genomics Institute in the US, to research the applications of CRISPR-Cas9 in crops and engineering microbiomes and address the question: “Can CRIPSR-Cas9 improve crop yields and nurture a balanced ecosystem?”. Details of potential applications in crops can be found from about half-way through the article (under the subheading: “Crops for the developing world”).
• For more in-depth scientific coverage of current CRISPR-Cas9 applications in plant and crop sciences, see Schiml and Puchta (2016) and/or Song et al. (2016).
• See also this overview in Nature "CRISPR: gene editing is just the beginning - The real power of the biological tool lies in exploring how genomes work"
• In November 2015, Sweden became the first country within the EU to deliver a legislative ruling on whether CRISPR-Cas9-editted plants fall within the definition of GMOs, and would subsequently be subject to the same restrictions as GMOs. The Swedish Board of Agriculture ruled that some plants edited using CRISPR-Cas9 technology do and others do not fall within the GMO definition. The distinction is based on the presence or absence of foreign DNA: plants with foreign DNA introduced through any means (CRISPR-Cas9 or otherwise) were ruled to be GMOs, while plants edited with CRISPR-Cas9 but lacking foreign DNA were ruled not to be. Read about the decision here and here.
• In 2016, Monsanto became the first agricultural business to receive a (non-exclusive) license for CRISPR-Cas9 technology, with some notable regulatory restrictions. Stat provides a helpful overview of what Monsanto can and can’t do with CRISPR-Cas9 technology. In short: they cannot use the technology to produce commercially available “terminator” seeds (which Monsanto do not produce commercially in any case, contrary to popular belief), nor to produce gene drives, nor may they use the technology in tobacco destined for sale for smoking. 

We hope that this has been an informative and useful read for FCRN members. If you have any questions or comments on this topic, please post them in the comments. We’d also be interested in hearing from anyone who is working on/with CRISPR: how are you using or investigating CRISPR and what do you envisage the outcomes of your work being? Please also feel free to share any further resources which you find useful or interesting on this topic.

Image credit: Top photo: ALL pneus: blé vert, Flickr, Creative commons licence 2.0 . Figure 1: ABC News "Precision genome editing with CRISPR-Cas9”