Genome editing: an introduction to CRISPR/Cas9

Damiano Martignago

Dr Damiano Martignago, Rothamsted Research

This week’s blog post was written by Dr Damiano Martignago, a genome editing specialist at Rothamsted Research.

 

Genome editing technologies comprise a diverse set of molecular tools that allow the targeted modification of a DNA sequence within a genome. Unlike “traditional” breeding, genome editing does not rely on random DNA recombination; instead it allows the precise targeting of specific DNA sequences of interest. Genome editing approaches induce a double strand break (DSB) of the DNA molecule at specific sites, activating the cell’s DNA repair system. This process could be either error-prone, thus used by scientists to deactivate “undesired” genes, or error-free, enabling target DNA sequences to be “re-written” or the insertion of DNA fragments in a specific genomic position.

The most promising among the genome editing technologies, CRISPR/Cas9, was chosen as Science’s 2015 Breakthrough of the Year. Cas9 is an enzyme able to target a specific position of a genome thanks to a small RNA molecule called guide RNA (gRNA). gRNAs are easy to design and can be delivered to cells along with the gene encoding Cas9, or as a pre-assembled Cas9-gRNA protein-RNA complex. Once inside the cell, Cas9 cuts the target DNA sequence homologous to the gRNAs, producing DSBs.

CRISPR/Cas9

The guide RNA (sgRNA) directs Cas9 to a specific region of the genome, where it induces a double-strand break in the DNA. On the left, the break is repaired by non-homologous-end joining, which can result in insertion/deletion (indel) mutations. On the right, the homologous-directed recombination pathway creates precise changes using a supplied template DNA. Credit: Ran et al. (2013). Nature Protocols.

 

Genome editing in crops

Together with the increased data availability on crop genomes, genome editing techniques such as CRISPR are allowing scientists to carry out ambitious research on crop plants directly, building on the knowledge obtained during decades of investigation in model plants.

The concept of CRISPR was first tested in crops by generating cultivars that are resistant to herbicides, as this is an easy trait to screen for and identify. One of the first genome-edited crops, a herbicide-resistant oilseed rape produced by Cibus, has already been grown and harvested in the USA in 2015.

Wheat powdery mildew

Researchers used CRISPR to engineer a wheat variety resistant to powdery mildew (shown here), a major disease of this crop. Image credit: NY State IPM Program. Used under license: CC BY 2.0.

 

Using CRISPR, scientists from the Chinese Academy of Sciences produced a wheat variety resistant to powdery mildew, one of the major diseases in wheat. Similarly, another Chinese research group exploited CRISPR to produce a rice line with enhanced rice blast resistance that will help to reduce the amount of fungicides used in rice farming. CRISPR/Cas9 has also been already applied to maize, tomato, potato, orange, lettuce, soybean and other legumes.

Genome editing could also revolutionize the management of viral plant disease. The CRISPR/Cas9 system was originally discovered in bacteria, where it provided them with molecular immunity against viruses, but it can also be moved into plants. Scientists can transform plants to produce the Cas9 and gRNAs that target viral DNA, reducing virus accumulation; alternatively, they can suppress those plant genes that are hijacked by the virus to mediate its own diffusion in the plants. Since most plants are defenseless against viruses and there are no chemical controls available for plant viruses, the main method to stop the spread of these diseases is still the destruction of the infected plant. For the first time in history, scientists have an effective weapon to fight back against plant viruses.

Cassava brown streak disease

The cassava brown streak disease virus can destroy cassava crops, threatening the food security of the 300 million people who rely on this crop in Africa. Image credit: Katie Tomlinson (for more on this topic, read her blog here).

 

Genome editing will be particularly useful in the genetic improvement of many crops that are propagated mainly by vegetative reproduction, and so very difficult to improve by traditional breeding methods involving crossing (e.g. cassava, banana, grape, potato). For example, using TALENs, scientists from Cellectis edited a potato line to minimize the accumulation of reducing sugars that may be converted into acrylamide (a possible carcinogen) during cooking.

 

Concerns about off-targets

One of the hypothesized risks of using CRISPR/Cas9 is the potential targeting of undesired DNA regions, called off-targets. It is possible to limit the potential for off-targets by designing very specific gRNAs, and all of the work published so far either did not detect any off-targets or, if detected, they occurred at a very low frequency. The number of off-target mutations produced by CRISPR/Cas9 is therefore minimal, especially if compared with the widely accepted random mutagenesis of crops used in plant breeding since the 1950s.

 

GM or not-GM

Genome editing is interesting from a regulatory point of view too. After obtaining the desired heritable mutation using CRISPR/Cas9, it is possible to remove the CRISPR/Cas9 integrated vectors from the genome using simple genetic segregation, leaving no trace of the genome modification other than the mutation itself. This means that some countries (including the USA, Canada, and Argentina) consider the products of genome editing on a case-by-case basis, ruling that a crop is non-GM when it contains gene combinations that could have been obtained through crossing or random mutation. Many other countries are yet to issue an official statement on CRISPR, however.

Recently, scientists showed that is possible to edit the genome of plants without adding any foreign DNA and without the need for bacteria- or virus-mediated plant transformation. Instead, a pre-assembled Cas9-gRNA ribonucleoprotein (RNP) is delivered to plant cells in vitro, which can edit the desired region of the genome before being rapidly degraded by the plant endogenous proteases and nucleases. This non-GM approach can also reduce the potential of off-target editing, because of the minimal time that the RNP is present inside the cell before being degraded. RNP-based genome editing has been already applied to tobacco plants, rice, and lettuce, as well as very recently to maize.

In conclusion, genome editing techniques, and CRISPR/Cas9 in particular, offers scientists and plant breeders a flexible and relatively easy approach to accelerate breeding practices in a wide variety of crop species, providing another tool that we can use to improve food security in the future.
For more on CRISPR, check out this recent TED Talk from Ellen Jorgensen:


About the author

Dr Damiano Martignago is a plant molecular biologist who graduated from Padua University, Italy, with a degree in Food Biotechnology in 2009. He obtained his PhD in Biology at Roma Tre University in 2014. His experience with CRISPR/Cas9 began in the lab of Prof. Fabio Fornara (University of Milan), where he used CRISPR/Cas9 to target photoperiod genes of interest in rice and generate mutants that were not previously available. He recently moved to Rothamsted Research, UK, where he works as Genome Editing Specialist, transferring CRISPR/Cas9 technology to hexaploid bread wheat with the aim of improving the efficiency of genome editing in this crop. He is actively involved with AIRIcerca (International Association of Italian Scientists), disseminating and promoting scientific news.

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Aquaporins capable of functioning as all-in-one osmotic systems

Caitlin Byrt

Dr Caitlin Byrt, University of Adelaide

This week’s post was written by Dr Caitlin Byrt, University of Adelaide, whose research focuses the roles of water-channeling proteins – aquaporins – and ion transport in plants.

 

Aquaporins are water-channel proteins that move water molecules through cell membranes. They are found in every kingdom of life. Cell membranes are semi-permeable to water, but often require more rapid movements of water across membranes; cells achieve this using aquaporins.

Aquaporins play key roles in your kidneys, which typically filter each of the three liters of plasma in your body 60 times per day – that’s 180 liters of plasma each day! Around three times your body weight in water passes through your own aquaporins each day.

Water on leaf

Around 50% of global rainfall passes through plants, and half of this moves through the aquaporins. Image credit: Dennis Seiffert. Used under license: CC BY-ND 2.0.

Aquaporin function

Have you got on the scales recently? Nearly 70% of your body weight is water. Water is the major component of cells in all of your tissues and this is the same for plants. Around 50% of global precipitation passes through plants, and half of this moves through aquaporins, so aquaporins account for the largest movement of mass for any protein on earth.

Often, in cell membranes, four aquaporin proteins will come together to form a tetramer to assist with the transportation of water across the cell membrane. There are types of aquaporins that only transport water, and others that transport glycerol, neutral acids or gasses. Historically, plant science literature has reported that the molecular structure of aquaporins prevents any charged particles, such as ions, from permeating. This is different in the animal world where there are reports of aquaporins that are permeable to ions. For example, in humans one of the most highly expressed aquaporins, AQP1, can function as a dual water and ion channel.

 

Testing plant aquaporins in frog cells

Recently, we observed that one of the most highly expressed plant aquaporins is permeable to ions when expressed in heterologous systems such as Xenopus laevis (frog) oocyte (egg) cells or yeast cells. This indicates that plants may also have types of aquaporins that can function as a dual water:ion channels.

 

Xenopus oocytes

The function of plant aquaporins can be studied by expressing them in different systems such as the Xenopus laevis oocyte cells pictured here. Photo credit: Dr Caitlin Byrt.

 

If you want to know if a particular plant aquaporin can function as a water channel you can test it by expressing the aquaporin in a laboratory oocyte expression system. We use a tiny needle to inject RNA coding for plant aquaporins of interest into the oocyte, and for control oocytes we inject the same amount of water. The oocytes are kept in a saline solution and we usually study them one or two days after injecting the RNA to allow time for them to synthesize the protein.

If you place oocytes expressing an aquaporin into water alongside control oocytes, then the aquaporin-expressing oocytes will burst much quicker than the controls because water rushes in through the aquaporin and causes the cell to swell rapidly. To explore whether a protein conducts ions, we use electrodes to measure the currents generated when charged ions pass across the oocyte membrane. We can also use ion-specific electrodes to explore which ions are transported.

 

AtPIP2;1 can transport water and ions

The plant aquaporin we studied is coded in the genome of the model plant Arabidopsis; it is a plasma membrane-located protein called AtPIP2;1. The AtPIP2;1 protein is known to be highly prevalent in root epidermal cell membranes, and it also functions in the guard cells of leaves, which act like tiny valves to regulate the uptake of carbon dioxide for photosynthesis and the release of water vapor.

 

AtPIP2 Arabidopsis

The model plant Arabidopsis has an aquaporin, AtPIP2;1, that can function as a dual water:ion channel. Photo credit: Dr. Jiaen Qiu.

 

We observed that AtPIP2;1 expression induces both water and ion (salt) movement across the cell membrane of oocytes. We know that the ionic conductance can be carried in part by sodium ions and that it is inhibited by calcium, cadmium and protons. This means AtPIP2;1 is a candidate for a previously reported calcium-sensitive non-selective cation channel responsible for sodium ion entry into Arabidopsis roots in saline conditions.

We are investigating the physiological role of ion permeable aquaporins in plants, and exploring how plants regulate the coupling of ion and water flow across key membranes. The regulation of ion permeability through plant aquaporins could be important in the control of water flow and regulation of cell volume. There is increasing discussion around the hypothesis that plants could drive water transport in the absence of water potential differences using salt and water co-transport, and this makes us wonder whether ion-permeable aquaporins may be involved. Testing whether ion-permeable aquaporins can function as an ‘all-in-one’ osmotic system in plants is an exciting new direction for research in this field.

 

Caitlin Byrt and Steve Tyerman

Dr. Caitlin Byrt, Professor Steve Tyerman and colleagues are investigating whether aquaporins permeable to ions are present in a range of different plant species. Photo credit: Wendy Sullivan

 

More information:

Byrt, C.S., Zhao, M., Kourghi, M., Bose, J., Henderson, S.W., Qiu, J., Gilliham, M., Schultz, C., Schwarz, M., Ramesh, S.A., Yool, A., and Tyerman, S.D., 2016. Non‐selective cation channel activity of aquaporin AtPIP2; 1 regulated by Ca2+ and pH. Plant, Cell & Environment.

Yool, Andrea J., and Alan M. Weinstein. New roles for old holes: ion channel function in aquaporin-1. Physiology 17.2 (2002): 68-72.

Battening down the hatches: priming plant defense

This week’s blog post was written by Dr. Mike Roberts (Lancaster University, UK).

Dr. Mike Roberts

Dr. Mike Roberts, Lancaster University, UK, Mike with an experiment to test the effects of parental herbivory on defense priming in the next generation of Arabidopsis plants. (Photo credit: Lancaster University)

It is widely accepted that achieving agricultural sustainability means reducing our reliance on synthetic agrochemicals. One major group of agrochemicals is pesticides, which include the insecticides and fungicides that protect crop plants against pests and diseases. Pests and diseases aren’t going to go away, so reducing pesticide usage means that alternative crop protection approaches are needed. EU Directive 2009/128/EC (Sustainable Use of Pesticides) recommends the use of integrated pest and disease management (IPM) – the combined use of multiple approaches that together provide sufficient protection.

Our contribution to this challenge has been to identify ways in which we might enhance a plant’s own natural defense mechanisms. Plants have a wide array of structural and chemical defenses that they can employ to fight off enemies. Many of these are inducible, meaning that they are only activated in response to attack, which allows plants to balance the costs and benefits of defense. For crop plants, these costs can often translate into reduced yields. Spraying with compounds that switch on inducible defenses, such as the plant hormones jasmonic acid (JA) and salicylic acid (SA), can make plants more resistant, but this approach also risks unwanted growth reductions.

The JA team

(Left to right) Jane Taylor, Mike Roberts and Nigel Paul, who led the research on defense priming using seed treatments, in the glasshouse at the Lancaster Environment Center. (Photo credit: Lancaster University)

Fortunately, evolution has produced another way of regulating inducible defenses that we can take advantage of: the phenomenon we refer to as ‘priming’. When we ourselves get infected with something like a virus, our immune system generates antibodies to quickly fight it off, but it also produces memory cells that can respond to the same infection many months, or even years, in the future, with a more rapid and effective immune response. This is the basis of the familiar concept of vaccination. While plants don’t make antibodies, they are nevertheless able to alter future patterns of defense activation in response to previous infection by disease or feeding by herbivorous insects; thus, priming results in a faster and stronger activation of future inducible defense responses.

 

Benefits of plant defense priming

Transgenerational immune priming enhances disease resistance in Arabidopsis. All the plants in the photograph were inoculated by spraying the whole tray with a suspension of Pseudomonas syringae bacteria. The plants on the right side of the photo are from seed collected from parent plants that were infected with the same P. syringae bacteria, whilst those on the left come from healthy parents. (Photo credit: Belinda Ameyaw)

 

If we can find ways to prime defenses in crop plants, we might be able to improve pest and disease resistance with minimal impacts on yield. One way to do this is through seed treatments. We found that treating seeds with the defense hormone JA provides long-term enhanced resistance against herbivory and some fungal diseases, without affecting growth and development. We were able to patent this discovery, and the approach has since been successfully commercialized. The same approach can also be used to prime defenses against other forms of biotic and abiotic stress.

Seed treatment

Tomato seeds being treated with jasmonic acid solution. (Photo credit: Lancaster University)

How and why seed treatments provide long-term defense priming can be explained by the phenomenon of transgenerational immune priming, which my lab has also been investigating. After our success with the seed treatment, we wondered, “What if seeds were exposed to hormones like JA during the course of their development on the parent plant?” We tested this by infecting plants with bacteria or exposing them to herbivores, and then examined defense in their offspring. Remarkably, we saw that priming responses established in the parent were passed on to the offspring; something we refer to as transgenerational immune priming.

The evidence we have at present suggests that the mechanism for this heritable stress memory is epigenetic, meaning the genes that control priming are chemically tagged to alter their activity. These epigenetic modifications don’t involve changes in the DNA sequence and are reversible, allowing rapid, flexible responses to environmental stress. Understanding the nature of these epigenetic changes may provide another way to exploit priming for crop protection. Introducing the right epigenetic marks onto genes in elite crop varieties may enable the priming of defense without altering their genetic make-up. Given the difficulty of introducing new chemical and biological methods of crop protection, which require time-consuming and costly regulatory approval before they can be brought to market, this could prove an especially attractive option in the future.

Does Australia hold the key to food security?

This article is reposted from the Devex blog with kind permission from the author, Lisa Cornish.

CIAT research

Plant samples in the genebank at the International Center for Tropical Agriculture’s Genetic Resources Unit, at the institution’s headquarters in Colombia. Credit: Neil Palmer / CIAT. Used under license: CC BY-SA 2.0.

It was too dry in the Australian region of Wimmera to produce crops last summer. This year, floods are set to wipe out yields again. Like a number of other regions across the planet, climate change is starting to be felt.

“It’s like this every year somewhere,” said Sally Norton, head of the Australian Grains Genebank, which stores diverse genetic material for plant breeding and research.

For Norton and many of her colleagues in agricultural genetics, the picture is increasingly clear: The variety of crops used today are not able to withstand the changing conditions and changes expected in the future.

Australia’s biodiversity may offer some help, according to discussions at the recent International Genebank Managers Annual General Meeting held in Horsham, Victoria. The gathering, which brings together 11 countries, focused on how to better conserve seeds, build databases to manage collections, boost capacity across the world and fill gaps in genebanks.

Researchers are particularly interested in crop wilds, “the ancestors of our domesticated crops,” Marie Haga, executive director of the The Crop Trust, explained to Devex. Australia is one of the richest sources of these seeds. “It’s like the wolf being the ancestor to our domesticated dogs. Crop wild relatives have traits that we have lost in the domestication process — they might need less water, might live in unfriendly conditions, may be resistant to pests and diseases.”

As climate change continues to batter agricultural yields, crop wild relatives could provide resilience. The seeds give breeders and farmers new options of plant varieties with traits to withstand a variety of conditions based on the harsh climates they are found — drought, fire, flood, poor soil, high salinity.

For Haga, crop wild relatives are a solution for food security. “The challenge is that many of the varieties widely used in modern agriculture are very vulnerable, because we have been breeding on the same line and they are adapted to very specific environment,” Haga said. Varieties that flourish today, she said, could wither as the climate fluctuates.

“Utilization of the natural diversity of crops is key to the future,” she said. “The climate is rapidly changing and we need to feed a growing population with more nutritious food. It is very hard to see how we can do this unless we go back to the building blocks of agriculture.”

Norton agreed: “Crop wild relatives have an amazing adaptability to changing conditions,” she told Devex. “When we talk about food security, we are talking about getting varieties in farm paddocks that have greater resilience to extreme conditions. It may not be the highest yield, but you are going to get something from this crop.”

Why have they been overlooked?

Crop wild relatives have so far been underutilized in the research and breeding process of crops.

“We have this fabulous natural diversity out there including 125,000 varieties of wheat and 200,000 varieties of rice.” Haga said. “We have not at all unlocked the potential of these crops.”

One reason is a dearth of research. “Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives,” a 10-year project led by Haga to ensure long-term conservation of crop wild relatives, conducted a global survey of distribution and conservation and found that of 1,076 known wild relatives for 81 crops, more than 95 percent are insufficiently represented in genebanks and 29 percent are completely missing. They are missing purely due to the fact that they have yet to be collected.

“Genebank managers are generally open to include crop wild relatives in their collections.” Haga said. “It’s just quite simply that not enough work has been done in this area and the full potential is yet to be realized,” she said.

At the moment, seeds are being collected in 25 countries around the world as part of the crop wild relative project, but it is Australia that has been identified as one of the richest sources for crop wild relatives in the world. Because of the continent’s low population density and vast, undisturbed natural environment, a wide variety of species have been conserved, said Norton.

Australia holds significant diversity of wild relatives of rice, sorghum, pigeon pea, banana, sweet potato and eggplant currently missing from global collections, according to research by the Australian Seed Bank Partnership. Forty species have been prioritized for collection with high hopes that they will enable crops to withstand the harsh environmental conditions in which Australian species are found.

There are still many areas of Australia yet to be surveyed, and the full extent of its agricultural riches may yet to be tapped.

Australian researchers will play an important role in pre-breeding local species of wild relatives to improve their use in breeding programs. Crop wild relatives have historically been used in a variety of crops including synthetic wheat, but Australian native wild relatives have been harder to include in the breeding process.

“In the next 10 to 15 years it would be surprising if there is not something coming out that hasn’t got a component of Australian native wild relative in it,” Norton said who is currently involved in the collection of Australian crop wild relatives.

Collection of crop wild relatives is time sensitive

There is an urgency to collect crop wild relatives. Not only are wild species needed now to support changing environmental conditions affecting crops and farming, urbanization is putting crop wild relatives at risk of disappearing.

“We need to collect these sooner rather than later,” Norton told Devex. “Urbanization has a big impact on any native environment, let alone crop wild relatives. We know what species on our target list are more threatened than others — urbanization, flooding and fire are all risks to their security. We certainly have a priority list of species to collect and we need to make sure we target the ones that are under threat first.”

Once the varieties are conserved, breeders and farmers will need to be convinced to start using crop wild relatives. Many are already on board. “Most breeders understand these wild relatives have great potential,” Haga said.

Still, wild relatives can be difficult to work with and produce a lower yield. Haga expects there to be some reluctance, though limited.

“The understanding of the need is increasing and we feel very confident that this material will be used and some of them may be the game changer we are looking for,” she said.

The plans for crop wild relatives

Haga’s 10-year project on crop wild relatives is halfway complete. They are nearing the end of the collection phase and entering the pre-breeding process, before they are able to breed and deliver new species to farmers.

Australian support for the program includes an agreement for additional amount of $5 million. That comes on top of previous support of $21.2 million to the Crop Diversity Endowment Fund, which supports crop diversity globally and with a focus on the Indo-Pacific. Brazil, Chile, Germany, Japan, New Zealand, Norway, Switzerland and the United States are among other supporters of the endowment fund that hopes to reach $850 million. In Australia, further resources are still required to fund and support better seed collection at home.

Globally, plans for crop wild relatives includes raising greater awareness of their potential and importance.

“We have a big job to do to create awareness of the important of crop diversity generally and crop wild relatives specifically,” Haga said. “We have been speaking for years about biodiversity in birds and fish and a range of other animals, but we have talked very little about conserving the diversity of crops. I will fight for all types of diversity, but especially plants.”


 

This article is reposted from the Devex blog with kind permission from the author, Lisa Cornish.