Many small regulatory elements, including miRNAs, miRNA binding sites, and cis-acting elements, comprise only 5~24 nucleotides and play important roles in regulating gene expression, transcription and translation, and protein structure, and thus are promising targets for gene function studies and crop improvement.
A protein hijacked from a bacterial pathogen helps to facilitate more precise genome editing in plants. A new genome editing system enhances the efficiency of an error-free DNA repair pathway, which could help improve agronomic traits in multiple crops.
Few technologies have made as big a splash in recent years as CRISPR/Cas9, and rightfully so. CRISPR/Cas9, or clustered regularly interspaced palindromic repeats (CRISPR) and associated genes, is a bacterial gene editing toolbox that allows researchers to edit genomic sequences much more precisely and efficiently than previously possible, opening up doors to new ways of doing research. As with many new biotechnologies, the application of CRISPR in biology began with genetic model organisms such as Arabidopsis thaliana. In recent research authors review the prospects for expanding the use of CRISPR for research beyond genetic model plant species.
Field trials show that poplar trees can be genetically modified to reduce negative impacts on air quality while leaving their growth potential virtually unchanged
Researchers have developed a set of tools that make it faster and easier to modify large segments of DNA. The new tools are for use in a technique called recombineering.
New research has found that the European Union’s opposition to modern crop breeding is at odds with the majority of other countries around the world and could jeopardise international trade.
At July’s New Breeding Technologies workshop held in Gothenburg, Sweden, Dr. Staffan Eklöf, Swedish Board of Agriculture, gave us an insight into their analysis of European Union (EU) regulations, which led to their interpretation that some gene-edited plants are not regulated as genetically modified organisms. We speak to him here on the blog to share the story with you.
Could you begin with a brief explanation of your job, and the role of the Competent Authority for GM Plants / Swedish Board of Agriculture?
I am an administrative officer at the Swedish Board of Agriculture (SBA). The SBA is the Swedish Competent authority for most GM plants and ensures that EU regulations and national laws regarding these plants are followed. This includes issuing permits.
You reached a key decision on the regulation of some types of CRISPR-Cas9 gene-edited plants. Before we get to that, could you start by explaining what led your team to start working on this issue?
It started when we received questions from two universities about whether they needed to apply for permission to undertake field trials with some plant lines modified using CRISPR/Cas9. The underlying question was whether these plants are included in the gene technology directive or not. According to the Swedish service obligation for authorities, the SBA had to deliver an answer, and thus had to interpret the directive on this point.
Could you give a brief overview of Sweden’s analysis of the current EU regulations that led to your interpretation that some CRISPR-Cas9 gene-edited plants are not covered by this legislation?
The following simplification describes our interpretation pretty well; if there is foreign DNA in the plants in question, they are regulated. If not, they are not regulated.
Our interpretation touches on issues such as what is a mutation and what is a hybrid nucleic acid. The first issue is currently under analysis in the European Court of Justice. Other ongoing initiatives in the EU may also change the interpretations we made in the future, as the directive is common for all member states in the EU.
CRISPR-Cas9 is a powerful tool that can result in plants with no trace of transgenic material, so it is impossible to tell whether a particular mutation is natural. How did this influence your interpretation?
We based our interpretation on the legal text. The fact that one cannot tell if a plant without foreign DNA is the progeny of a plant that carried foreign DNA or the result of natural mutation strengthened the position that foreign DNA in previous generations should not be an issue. It is the plant in question that should be the matter for analysis.
Does your interpretation apply to all plants generated using CRISPR-Cas9, or a subset of them?
It applies to a subset of these gene-edited plants. CRISPR/Cas9 is a tool that can be used in many different ways. Plants carrying foreign DNA are still regulated, according to our interpretation.
What does your interpretation mean for researchers working on CRISPR-Cas9, or farmers who would like to grow gene-edited crops in Sweden?
It is important to note that, with this interpretation, we don’t remove the responsibility of Swedish users to assess whether or not their specific plants are included in the EU directive. We can only tell them how we interpret the directive and what we request from the users in Sweden. Eventually I think there will be EU-wide guidelines on this matter. I should add that our interpretation is also limited to the types of CRISPR-modified plants described in the letters from the two universities.
We are currently waiting for the EU to declare whether CRISPR-Cas9 gene-edited plants will be regulated in Europe. Have policymakers in other European countries been in contact with you regarding Sweden’s decision process?
Yes, there is a clear interest; for example, Finland handled a very similar case. Other European colleagues have also shown an interest.
What message would you like plant scientists to take away from this interview? If you could help them to better understand one aspect of policymaking, what would it be?
Our interpretation is just an interpretation and as such, it is limited and can change as a result of what happens; for example, what does not require permission today may do tomorrow. Bear this in mind when planning your research and if you are unsure, it is better to ask. Moreover, even if the SBA (or your country’s equivalent) can’t request any information about the cultivation of plants that are not regulated, it is good to keep us informed.
I think it is vital that legislation meets reality for any subject. It is therefore good that pioneers drive us to deal with difficult questions.
This week’s blog was written by Dr Craig Cormick, the Creative Director of ThinkOutsideThe. He is one of Australia’s leading science communicators, with over 30 years’ experience working with agencies such as CSIRO, Questacon and Federal Government Departments.
So what do you think CRISPR cabbage might taste like? CRISPR-crispy? Altered in some way?
Participants at the recent Society for Experimental Biology/Global Plant Council New Breeding Technologies workshop in Gothenburg, Sweden, had a chance to find out, because in Sweden CRISPR-produced plants are not captured by the country’s GMO regulations and can be produced.
Professor Stefan Jansson, one of the workshop organizers, has grown the CRISPR cabbage (discussed in his blog for GPC!) and not only had it included on the menu of the workshop dinner, but also had samples for participants to take away. Some delegates were keen to pick up the samples while others were unsure how their own country’s regulatory rules would apply to them
— Wayne Parrott (@ProfParrott) July 7, 2017
The uncertainty some delegates felt about the legality of taking a CRISPR cabbage sample home was a good demonstration of the diversity of regulations that apply – or may apply – to new breeding technologies, such as CRISPR and gene editing – and there was considerable discussion at the workshop on how European Union regulations and court rulings may play out, affecting both the development and export/import of plants and foods produced by the new technologies.
A lack of certainty has meant many researchers are unable to determine whether their work will need to be subjected to costly and time-consuming regulations or not.
The need for new breeding technologies was made clear at the workshop, which was attended by 70 people from 17 countries, with presentations on the need to double our current food production to feed the world in 2050 and reduce crop losses caused by problems such as viruses, which deplete crops by 10–15%.
The two-day workshop, held in early July, looked at a breadth of issues, including community attitudes, gene editing success stories, and tools and resources. But discussions kept coming back to regulation.
Regulations of gene technologies were largely developed 20 years ago or so, for different technologies than now exist, and as a result are not clear enough for researchers to determine whether different gene editing technologies they are working on may be governed by them or not.
The diversity of regulations is also going to be an issue, for some countries may allow different gene editing technologies, but others may not allow products developed using them to be imported.
That led to the group beginning to develop a statement that captured the feeling of the workshop, which, when complete, it is hoped will be adopted by relevant agencies around the world to develop their own particular positions on gene editing technologies. It would be a huge benefit to have a coherent and common line in an environment of mixed regulations in mixed jurisdictions.
And as to the initial question of what CRISPR cabbage tastes like – just like any cabbage you might buy at your local supermarket or farmers market, of course – since it is really no different.
— GARNet (@GARNetweets) July 7, 2017
Could you begin by giving our readers a brief overview of synthetic biology?
Synthetic biology involves the application of engineering principles to biological systems. One approach to understanding a biological system is to break it down into smaller parts, which can be used to design new properties. These redesigned pieces can be reassembled into a new system, tested experimentally, and refined in an iterative process. Synthetic biology projects that are underway in our lab include designing plastids such as chloroplasts with new metabolic functions, and in the longer term the design and assembly of synthetic chloroplast genomes.
Why do you use chloroplasts for synthetic biology systems?
Chloroplasts have a relatively small genome, coding for about 100 genes. Importantly, exogenous (foreign) genes coding for new functions can be precisely introduced into the chloroplast genome. All of the plastids within a plant contain the same genome so, once established, the user-designed reprogrammed plastids will be present throughout the plant. Chloroplasts can also produce very high levels of protein; researchers have achieved expression levels where over 70% of the total soluble protein in the leaves is the engineered protein. Expression in tomato fruit is also possible.
Multiple genes can be introduced into chloroplasts and expressed coordinately, allowing the metabolic engineering of more complex processes. The upper size limit for insertions is not known but is likely to be above the 50,000 nucleotide insertion achieved to date. Furthermore, chloroplasts and other plastids are important metabolic hubs and contain a wide variety of chemical substrates useful for metabolic engineering.
Could you describe the current state of our ability to engineer chloroplasts?
Chloroplast engineering is routine in many labs around the globe. Although there are multiple chloroplasts in every cell, the process of converting all the chloroplasts to a single population of engineered genomes is not an issue. Most researchers use the tobacco plant because it is easily transformed, but other crops are amenable to transformation, including oilseed rape, soybean, tomato, and potato (cereals such as rice and wheat are more problematic). There has been progress with developing the inducible expression of exogenous genes in chloroplasts too.
What challenges/differences do you face when transforming chloroplast genomes when compared to the nuclear genome?
Typical genetic modification of the DNA in the nucleus is performed by introducing exogenous genes in T-DNA. T-DNA is transferred to the plant using the bacterium Agrobacterium tumefaciens, which is an efficient process, but the T-DNA integrates ‘randomly’ at many sites within chromosomes and different lines can have variable expression levels due to positional effects and gene silencing.
A. tumefaciens-mediated gene delivery systems do not work for chloroplast transformation. Most chloroplast transformation labs introduce genes into plastids by blasting cells with gold or tungsten particles coated with DNA. Because chloroplast genomes are present in multiple copies per cell, the process of converting all resident chloroplasts to the transgenic genome requires a continued period of selection. This means that the isolation of chloroplast transformants can take slightly longer than nuclear transformation. In our lab, we speed up this process by using restoration of photosynthesis to select chloroplasts with exogenous genes. Once plants with a uniform population of transgenic plastid genomes have been isolated, the transgenes are stable and inherited through the maternal line.
For the novice, I would say nuclear transformation using A. tumefaciens is easier to accomplish than chloroplast transformation.
Last year you reported that chloroplasts degrade in mature sperm cells just prior to fertilization. Could you elaborate on how this might be utilized in future crop breeding?
Chloroplasts are inherited from the female parent in wheat. This is useful because it restricts the pollen-mediated spread of chloroplast-localized transgenes into the environment. Previously, no-one had studied the mechanism of maternal chloroplast inheritance in wheat using modern cell biology tools. With our collaborators Lucia Primavesi, Huixia Wu, and Huw Jones at Rothamsted Research, we developed an efficient method to observe small non-green plastids in wheat pollen in real time. We found that the plastids were destroyed during the maturation of sperm cells, which explained the absence of paternal plastids in the offspring.
This discovery has applications in crop breeding. Anther culture is a powerful technique where new homozygous plants can be produced by doubling the chromosome numbers of haploid plants regenerated from pollen. This technique has been challenging in cereals, as chloroplast degradation in pollen leads to a high percentage of albino plants (in some cases 100% albinos). Understanding how to prevent the destruction of plastids in pollen sperm cells will improve this technique in cereals, which could speed up crop breeding in the future.
What sorts of processes have you successfully transformed into chloroplasts, and what kinds of results have you achieved?
We have expressed a variety of exogenous genes in chloroplasts, from those conferring resistance to herbicides to vaccine epitopes and pharmaceutical proteins:
- Plants expressing the bar gene in chloroplasts were resistant to the herbicide glufosinate (also known as phosphinothricin).
- A chloroplast-expressed viral epitope was used to identify samples of human blood infected with the hepatitis C virus.
- Human transforming growth factor 3 (hTGFβ3), a potential wound healing drug, accumulated to high concentrations in chloroplasts, and could be processed to a pure active form resembling clinical grade hTGFβ3.
- In collaboration with Ray Dixon, Cheng Qi, and Mandy Dowson-Day at the John Innes Centre, we investigated the feasibility of introducing nitrogen-fixing genes into chloroplasts. This work was initiated in a unicellular green alga with the bacterial nifH gene.
What is the cutting edge of chloroplast transformation research?
Chloroplast genes are important for plant growth and development but they are difficult to improve by conventional breeding methods. We recently developed a method to edit plastid genomes, which allows beneficial single point mutations to be introduced into chloroplast genes. This is important because the resulting plants have an identical genome to the original cultivar apart the single base substitution, potentially leading to a new class of biotech crop.