Targeted gene knockouts in crops using RNA-guided Cas9 nuclease

Lead Research Organisation: John Innes Centre
Department Name: Crop Genetics

Abstract

In developing improved crops, plant breeders have always depended on finding variation or changes within a population to identify characteristics (or genetic traits) that could potentially offer an advantage to the crop. This variation is due to changes that can occur in the genetic sequence (DNA) of the crop plant over time - and is a normal part of evolution (mutations in the DNA). These changes can be responsible for many new characteristics such as plants with increased disease resistance or improved nutritional quality. Sometimes the desired variation is not available so breeders have used methods to introduce changes in the DNA such as chemical or radiation treatment of seed hoping to find the required variation in the resulting plants. These methods have been widely used in conventional plant breeding for decades. Changes to the DNA sequence often leads to a mutation that stops a particular gene from working. Mutations are incredibly useful as research tools as well as useful to plant breeders. If a scientist can find a mutation in the gene that they are interest in (i.e. generating a non-functional gene, or a gene knock-out) then they have a way of working out what that gene does.

Until recently, methods to introduce mutations were non-specific meaning they could target a large number of genes within the plant, hopefully including the DNA region, or gene, of interest. Now for the first time we have a technology to introduce mutations into a specific gene of interest. This is referred to as genome editing. A specific genome editing technology called CRISPR / Cas9 allows easy targeting of any gene of interest and we have demonstrated that this works well in both barley and Brassica oleracea (broccoli). In this project, we will offer to create plants with mutations in specific gene(s) of interest for the research community. This will allow more rapid progress in a range of research programmes. In addition, the resource we will offer could lead to plants with desirable characteristics to include in new crop varieties. As this technique is very new there have not yet been decisions as to how it should be regulated. The data that we will collect in this project will also help regulators to make decisions on future regulation of genome editing.

Technical Summary

The ability to introduce mutations in specific genes has long been a goal of plant scientists. Mutant lines have been a valuable resource for plant breeders and for the plant research community, however, availability in crop plants has to date been limited. Now, for the first time we have the ability to produce targeted mutations in any gene of interest using genome editing. Genome editing techniques all rely on a targeting strategy to deliver a nuclease to a specific genomic location where it causes a DNA break. Errors are often introduced during repair of the break introducing mutations. The first genome editing strategies required time-consuming and costly assembly of constructs to create synthetic DNA-binding proteins. However, the recently developed RNA-guided Cas9 system is much simpler requiring only two components, the Cas9 nuclease and a guide RNA. In this project we will provide a targeted mutagenesis resource for the crop research community based on RNA-guided Cas9 induced mutations. Within the BRACT crop transformation platform we have demonstrated the use of RNA-guided Cas9 to generate mutations in target genes in both barley and Brassica oleracea and have shown stable transmission of these mutations to progeny plants. In this project, we will allow researchers to request the production of knock-out mutants in their crop gene of interest free of charge. We will target 50 genes in barley and Brassica oleracea and deliver plants containing targeted mutations. In addition, we will respond to demand from the community and target another 10 genes in other crop species as required. Alongside the key targeted mutagenesis resource we will develop systems for the rapid screening of guide RNAs to ensure functionality and we will evaluate new methods for high-throughput analysis of transgenic plants for targeted mutations.

Planned Impact

Direct genome engineering technologies are widely anticipated to transform fundamental research in the near term. They also promise wider benefits to agricultural productivity as new traits are demonstrated in crops and feed through to breeding programmes. Our strategy is to facilitate the widest possible adoption of this key innovation from the plant science research community through to the agri-food sector.

There will be a range of beneficiaries from the proposed project. Firstly, it will give the academic community the opportunity to advance their research programmes in ways not previously possible by the provision of targeted mutations in their specific genes of interest. The resource will also be available to SMEs if they wish to access it for research purposes. This will allow them to evaluate the new technology and will provide data to inform future strategies for crop improvement. As the IP associated with the technology is complex, within the project we will commission a preliminary FTO search and opinion and this will enable us to provide information and advice on possible commercial exploitation. In addition, we will provide regulators, policy makers and government agencies with the latest data on the use of genome editing technologies to inform current debate, in particular within the EU, as to how the technology should be regulated. The data built up during the project will also contribute to the safety assessment of genome editing techniques.

Compared to genetic modification, genome editing can result in just a small mutation in a target gene and no other changes in the plant genome. This may benefit the wider public by helping allay fears over new plant breeding techniques due to the very precise nature of the technology. The wider public will also benefit as we plan to produce a video explaining genome editing in an accessible and engaging way. We also plan to engage with the media and potential end users of the technology; including breeders, farmers, food processors, retailers and consumer groups, to seek to inform a productive dialogue and to positively influence policy development in the UK. These activities will be supported by JIC's experienced communications and KEC teams.

The BRACT platform, in association with the JIC's in-house intellectual property agency, Plant Bioscience Ltd. (PBL), already has a strong and successful track record in providing crop transformation services to confirm gene function. The development of RNA-guided Cas9 based technologies will be a valuable addition to the portfolio of resources BRACT can offer allowing more innovations to move through patenting or licensing. Ultimately, the technology will contribute to the BBSRC's aspirations to develop the new approaches and technologies needed to support bioscience as it becomes more predictive and to accelerate the translation of research outputs into business applications, thus enhancing its social and economic impact.

Publications

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Eriksson D (2018) A Welcome Proposal to Amend the GMO Legislation of the EU. in Trends in biotechnology

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Harwood WA (2019) An Introduction to Barley: The Crop and the Model. in Methods in molecular biology (Clifton, N.J.)

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Hinchliffe A (2019) Agrobacterium-Mediated Transformation of Barley Immature Embryos. in Methods in molecular biology (Clifton, N.J.)

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Hundleby P (2018) Impacts of the EU GMO regulatory framework for plant genome editing in Food and Energy Security

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Lawrenson T (2019) Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9. in Methods in molecular biology (Clifton, N.J.)

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Lawrenson T (2019) Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9. in Methods in molecular biology (Clifton, N.J.)

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Raitskin O (2016) Multi-gene engineering in plants with RNA-guided Cas9 nuclease. in Current opinion in biotechnology

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Vazquez-Vilar M (2018) DNA assembly standards: Setting the low-level programming code for plant biotechnology. in Plant science : an international journal of experimental plant biology

 
Description A new improved methodology for the detection of mutations generated using CRISPR / Cas9. This method is based on PCR and direct sequencing. Data on the efficiency of genome editing over a larger number of targets has been generated.
Exploitation Route Our findings using CRISPR / Cas9 for generating targeted mutations have already contributed to new research proposals and are allowing faster progress in a number of research projects both internally and externally.
Sectors Agriculture, Food and Drink

 
Description The first call for target genes for this resource led to applications from 20 researchers for 40 target genes. This demonstrated the demand for the resource. We have started work on 20 target genes in barley and Brassica having accepted one target from each research group initially. Therefore with this first set of target genes we expect the resource to impact 20 different research areas. The second call for target genes led to applications from 46 individual researchers from 28 Universities / Research Institutes for 83 target genes. This gives a total number of requests for 126 target gene knock-outs, over double the number that the resource can cope with. With help from the advisory committee, we have selected 40 targets from this second call across a range of crops (wheat, barley, Brassica oleracea, Brassica napus and tomato). Work has just started on these new targets. Training has also been provided to one SME, one start-up company and to researchers from a number of labs.
First Year Of Impact 2016
Sector Agriculture, Food and Drink
Impact Types Economic,Policy & public services

 
Description Member of Advisory Committee on Animal Feedingstuffs (ACAF)
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Member of Global Plant Council New Breeding Technologies Working Group
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Description Breeders day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Talk and demonstration on genome editing during Breeders day
Year(s) Of Engagement Activity 2017
 
Description CBB4 conference Budapest 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Keynote presentation at the 4th Conference of Cereal Biotechnology and Breeding on CRISPR / Cas9 genome editing in wheat and barley held in Budapest.
Year(s) Of Engagement Activity 2017
 
Description CPSC PhD course: SYNthetic BIOlogy, From pro- to eukaryotic SYStems (SYNBIOSYS) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Delivered lecuryres and workshops as part of the University of Copenhagen's CPSC PhD course: SYNthetic BIOlogy, From pro- to eukaryotic SYStems (SYNBIOSYS)
Year(s) Of Engagement Activity 2017
URL http://cpsc.ku.dk/calendar/2017/cpsc-phd-summer-course-2017-synbiosys/
 
Description CRISPR AgBio meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation on CRISPR/Cas9 Genome Editing in UK Crops at AgBio conference. New contacts with industry and academics made.
Year(s) Of Engagement Activity 2017
 
Description GM technologies Interview for BBC Radio Scotland - Good Morning Scotland' programme 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Radio interview to discuss GM technologies, GM food and new gene editing techniques
Year(s) Of Engagement Activity 2017
 
Description Live television interview with Australian Broadcasting Corporation 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Live television interview with 'The World', international news and current affairs show (Australian Broadcasting Corporation). Received multiple enquiries and comments from academics and general public following broadcast.
Year(s) Of Engagement Activity 2017
URL http://www.abc.net.au/news/2017-01-26/the-year-of-crispr:-scientists-celebrate-new/8213772
 
Description Norwich Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Hands on activity at the Norwich Science Festival entitled 'The CRISPR Craze'. We had over 500 children and adults visit the stand over 2 days. Lots of discussion with members of the public about the technology.
Year(s) Of Engagement Activity 2017
 
Description Plant Genetics Resources and the Sustainable Development Goals: Needs, Rights, and Opportunities. Rockefeller Bellagio Center, Bellagio, Italy 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact 18 experts from a range of disciplines were hosted by The Rockefeller Foundation at its Bellagio Center in Italy to discuss how plant genetic resources relate to and can be instrumental in meeting the United Nations' Sustainable Development Goals (SDGs).

The group had three objectives, which were to:
• Advance the ongoing global dialogue about how science, technology, data, and digital information about plant genetic resources (PGR) may impact and potentially help agriculture adapt to the changing climate and augment food and nutritional security, particularly in the developing world
• In the context of the SDGs, explore the interaction between emerging R&D opportunities (emanating from scientific and technological advances and increased data sharing capability) and the current policy framework related to access, use, and benefit-sharing of PGR and related information.
• Identify pathways for harmonizing the PGR policy framework with rapid developments in science and information technology to ensure safeguards and enhance opportunities to address the SDGs.
The group agreed to pursue three outputs, to be completed in the next six months:
• As a group, the participants will develop a comprehensive meeting report for public distribution. This report was initiated during the meeting and will be completed early in 2017.
• During the meeting, participants from the Diversity Seek Initiative (DivSeek) gathered input for their response to an invitation by the Secretary of the International Treaty for Plant Genetic Resources in Food and Agriculture (ITPGRFA) "to report on the implications for the objectives of the Treaty of the technologies underlying the DivSeek initiative" (as per paragraph 6 of Resolution 3/2015 of the Sixth Session of the Treaty Governing Body). This outcome can be used by the Secretariat to compile a synthesis on this report for consideration by the Governing Body of the Treaty at its Seventh Session in 2017. Participants agreed to provide additional input on the draft submission to the Treaty upon request from DivSeek.
• Finally, at least one scholarly article will be published as a result of this meeting describing the issues discussed and exploring potential solutions and bottlenecks to progress. The group agreed that it would be ideal to aim to develop a balanced treatment such that all participants would be happy to be identified as authors or contributors where their expertise was relevant to the subject matter. Participants also discussed how best to communicate the results with other audiences and agreed to consult with one another if any developed blog posts or articles related to the meeting.
Year(s) Of Engagement Activity 2016
 
Description Presentation at Crop Transformation meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation entitled 'Transformation and Genome Editing in Wheat and Barley' at the 4th International Conference 'Plant Transformation & Biotechnology' in Vienna.
Year(s) Of Engagement Activity 2017
 
Description Radio interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Radio interviews on gene editing in plants
Year(s) Of Engagement Activity 2016,2017
 
Description SEB Plant Satellite meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk on the Brassica / barley gene editing resource at SEB meeting: New Breeding Technologies in the Plant Sciences - Applications and Implications
 
Year(s) Of Engagement Activity 2017
 
Description Seminar at the Triticeae Research Institute, Sichuan Agricultural University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation on 'From Genetic Modification to Genome Editing in wheat and barley' at Sichuan Agricultural University.
Year(s) Of Engagement Activity 2017
 
Description Undergraguate student conference in Portugal 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Presentation entitled Crop genome engineering - GM to genome editing at a meeting organised by students at Universidade de Trás-os-Montes e Alto Douro, Portugal
Year(s) Of Engagement Activity 2017