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Cas9 Archives - The Global Plant Council

Genome editing: an introduction to CRISPR/Cas9

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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.

Now That’s What I Call Plant Science 2015

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With another year nearly over we recently put out a call for nominations for the Most Influential Plant Science Research of 2015. Suggestions flooded in, and we also trawled through our social media feeds to see which stories inspired the most discussion and engagement. It was fantastic to read about so much amazing research from around the world. Below are our top five, selected based on impact for the plant science research community, engagement on social media, and importance for both policy and potential end product/application.

Choosing the most inspiring stories was not an easy job. If you think we’ve missed something, please let us know in the comments below, or via Twitter! In the coming weeks we’ll be posting a 2015 Plant Science Round Up, which will include other exciting research that didn’t quite make the top five, so watch this space!

  1. Sweet potato is a naturally occurring GM crop
Sweet potato contains genes from bacteria making it a naturally occurring GM crop

Sweet potato contains genes from bacteria making it a naturally occurring GM crop. Image from Mike Licht used under creative commons license 2.0

Scientists at the International Potato Center in Lima, Peru, found that 291 varieties of sweet potato actually contain bacterial genes. This technically means that sweet potato is a naturally occurring genetically modified crop! Alongside all the general discussion about GM regulations, particularly in parts of Europe where regulations about growing GM crops have been decentralized from Brussels to individual EU Member States, this story caused much discussion on social media when it was published in March of this year.

It is thought that ancestors of the modern sweet potato were genetically modified by bacteria in the soil some 8000 years ago. Scientists hypothesize that it was this modification that made consumption and domestication of the crop possible. Unlike the potato, sweet potato is not a tuber but a mere root. The bacteria genes are thought to be responsible for root swelling, giving it the fleshy appearance we recognize today.

This story is incredibly important, firstly because sweet potato is the world’s seventh most important food crop, so knowledge of its genetics and development are essential for future food supply. Secondly, Agrobacterium is frequently used by scientists to artificially genetically modify plants. Evidence that this process occurs in nature opens up the conversation about GM, the methods used in this technology, and the safety of these products for human consumption.

Read the original paper in PNAS here.

  1. RNA-guided Cas9 nuclease creates targetable heritable mutations in Barley and Brassica

Our number two on the list also relates to genetic modification, this time focusing on methods. Regardless of whether or not we want to have genetically modified crops in our food supply, GM is a valuable tool used by researchers to advance knowledge of gene function at the genetic and phenotypic level. Therefore, systems of modification that make the process faster, cheaper, and more accurate provide fantastic opportunities for the plant science community to progress its understanding.

The Cas9 system is a method of genome editing that can make precise changes at specific locations in the genome relatively cheaply. This novel system uses small non-coding RNA to direct Cas9 nuclease to the DNA target site. This type of RNA is small and easy to program, providing a flexible and easily accessible system for genome editing.

Barley in the field

Barley in the field. Image by Moldova_field used under creative commons license 2.0

Inheritance of genome modifications using Cas9 has previously been shown in the model plants, Arabidopsis and rice. However, the efficiency of this inheritance, and therefore potential application in crop plants has been questionable.

The breakthrough study published in November by researchers at The Sainsbury Laboratory and John Innes Centre both in Norwich, UK, demonstrated the mutation of two commercial crop plants, Barley and Brassica oleracea, using the Cas9 system and subsequent inheritance mutations.

This is an incredibly exciting development in the plant sciences and opens up many options in the future in terms of genome editing and plant science research.

Read the full paper in Genome Biology here.

  1. Control of Striga growth

Striga is a parasitic plant that mainly affects parts of Africa. It is a major threat to food crops such as rice and corn, leading to yield losses worth over 10 billion US dollars, and affecting over 100 million people.

Striga infects the host crop plant through its roots, depriving them of their nutrients and water. The plant hormone strigolactone, which is released by host plants, is known to induce Striga germination when host plants are nearby.

In a study published in August of this year the Striga receptors for this hormone, and the proteins responsible for striga germination were identified.

Striga plants are known to wither and die if they cannot find a host plant upon germination. Induction of early germination using synthetic hormones could therefore remove Striga populations before crops are planted. This work is vital in terms of regulating Striga populations in areas where they are hugely damaging to crop plants and people’s livelihoods.

Read the full study in Science here.

Striga, a parasitic plant. Also known as Witchweed.

Striga, a parasitic plant. Also known as Witchweed. Image from the International Institute of Tropical Agriculture used under creative commons license 2.0

  1. Resurrection plants genome harvesting

Resurrection plants are a unique group of flora that can survive extreme water shortages for months or even years. There are more than 130 varieties in the world, and many researchers believe that unlocking the genetic codes of drought-tolerant plants could help farmers working in increasingly hot and dry conditions.

During a drought, the plant acts like a seed, becoming so dry that it appears dead. But as soon as the rains come, the shriveled plant bursts ‘back to life’, turning green and robust in just a few hours.

In November, researchers from the Donald Danforth Plant Science Centre in Missouri, US, published the complete draft genome of Oropetium thomaeum, a resurrection grass species.

O. thomaeum is a small C4 grass species found in Africa and India. It is closely related to major food feed and bioenergy crops. Therefore this work represents a significant step in terms of understanding novel drought tolerance mechanisms that could be used in agriculture.

Read the full paper in Nature here.

  1. Supercomputing overcomes major ecological challenge

Currently, one of the greatest challenges for ecologists is to quantify plant diversity and understand how this affects plant survival. For the last 500 years independent research groups around the world have collected this diversity data, which has made organization and collaboration difficult in the past.

Over the last 500 years, independent research groups have collected a wealth of diversity data. The Botanical Information and Ecology Network (BIEN) are collecting and collating these data together for the Americas using high performance computing (HPC) and data resources, via the iPlant Collaborative and the Texas Advanced Computing Center (TACC). This will allow researchers to draw on data right from the earliest plant collections up to the modern day to understand plant diversity.

There are approximately 120,000 plant species in North and South America, but mapping and determining the hotspots of species richness requires computationally intensive geographic range estimates. With supercomputing the BIEN group could generate and store geographic range estimates for plant species in the Americas.

It also gives ecologists the ability to document continental scale patterns of species diversity, which show where any species of plant might be found. These novel maps could prove a fantastic resource for ecologists working on diversity and conservation.

Read more about this story on the TACC website, here.