A taste of CRISPR

Dr Craig CormickThis 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.

 

 

Regulatory issues

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.

Outdated regulations

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.

CRISPR cabbage

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.

 

Want to read more about CRISPR? Check out our interview with Prof. Stefan Jansson or our introduction to CRISPR from Dr Damiano Martignago.

Synthetic biology in chloroplasts

Dr Anil Day, University of Manchester

Dr Anil Day, University of Manchester

This week we spoke to Dr. Anil Day, a synthetic biologist at the University of Manchester who has developed an impressive array of tools and techniques to transform chloroplast genomes.

 

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.

 

Anil Day examines transformed plants

Dr. Anil Day examines a cabinet of transformed plants. Credit: Dr. Anil Day.

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.

Plastids in plants

Plants have several types of plastids, including green photosynthetic chloroplasts, pigment-containing chromoplasts, and starch-containing amyloplasts. Credit: Dr. Anil Day.

 

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.

 

Edited chloroplasts

A tobacco plant containing leaf areas with edited (pale green) and normal (darker green) chloroplasts. Credit: Dr. Anil Day.

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 PrimavesiHuixia 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.

 

Selection of transformed plants

Transformed plantlets are selected by their ability to survive on a herbicide-containing agar plate, and can then be grown up into mature plants. Credit: Dr. Anil Day.

 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.

Registration open for GPC/SEB New Breeding Technologies Workshop!

New Breeding Technologies in the Plant Sciences – Applications and Implications in Genome Editing

Gothenburg, Sweden, 7-8th July 2017

REGISTRATION FOR THIS MEETING IS NOW OPEN!

Organised by: Dr Ruth Bastow (Global Plant Council), Dr Geraint Parry (GARNet), Professor Stefan Jansson (Umeå University, Sweden) and Professor Barry Pogson (Australian National University, Australia).

Targeted genome engineering has been described as a “game-changing technology” for fields as diverse as human genetics and plant biotechnology. Novel techniques such as CRISPR-Cas9, Science’s 2015 Breakthrough of the Year, are revolutionizing scientific research, allowing the targeted and precise editing of genomes in ways that were not previously possible.

Used alongside other tools and strategies, gene-editing technologies have the potential to help combat food and nutritional insecurity and assist in the transition to more sustainable food production systems. The application and use of these technologies is therefore a hot topic for a wide range of stakeholders including scientists, funders, regulators, policy makers and the public. Despite its potential, there are a number of challenges in the adoption and uptake of genome editing, which we propose to highlight during this SEB satellite meeting.

One of the challenges that scientists face in applying technologies such as CRISPR-Cas9 to their research is the technique itself. Although the theoretical framework for using these techniques is easy to follow, the reality is often not so simple. This meeting will therefore explain the principles of applying CRISPR-Cas9 from experts who have successfully used this system in a variety of plant species. We will explore the challenges they encountered as well as some of the solutions and systems they adopted to achieve stably transformed gene-edited plants.

The second challenge for these transformative technologies is how regulatory bodies will treat and asses them. In many countries gene editing technologies do not fit within current policies and guidelines regarding the genetic modification and breeding of plants, as it possible to generate phenotypic variation that is indistinguishable from that generated by traditional breeding methods. Dealing with the ambiguities that techniques such as CRISPR-Cas9 have generated will be critical for the uptake and future use of new breeding technologies. This workshop will therefore outline the current regulatory environment in Europe surrounding gene editing, as well as the approaches being taken in other countries, and will discuss the potential implications and impacts of the use of genome engineering for crop improvement.

Overall this meeting will be of great interest to plant and crop scientists who are invested in the future of gene editing both on a practical and regulatory level. We will provide a forum for debate around the broader policy issues whilst include opportunities for in-depth discussion regarding the techniques required to make this technology work in your own research.

This meeting is being held as a satellite event to the Society for Experimental Biology’s Annual Main Meeting, which takes place in Gothenburg, Sweden, from the 3–6th July 2017.