Refining genome editing in livestock

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

This project aims to refine genetic engineering (GE) technology thus providing better tools for both the academic and commercial sectors working in the biosciences. GE livestock were first produced about 25 years ago. Since then improvements in the technology used have been developed. However, they remain inefficient and lack precision. The ideal technology for producing GE livestock would be one that enable the efficient a precise engineering of the zygote.

Excitingly this is now emerging - we can now engineer the zygote with designer hybrid endonucleases that do not leave a 'transgenic' mark. In essence these enzymes function as molecular scissors cutting the genome precisely where you want it to be cut. This emerging technology is based on genome editors, examples of which include ZFN and TALEN. We have recently demonstrated that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs.

We now aim to refine the method arguing that efficiency improvements are possible for GE technology in livestock, focussing on the following four objectives which build on our preliminary data: increase efficiency, reducing variability, broadening application through developing ability to change specific DNA sequences, and demonstrating the method in ruminants.

This technology has the potential to be transformative in its impact if applied in the agricultural and biotechnology sectors.

Technical Summary

We have recently demonstrated that both TALEN and ZFN DNA editors injected directly into pig zygotes can produce live genome edited pigs. This novel achievement paves the way for precise genome engineering of livestock independent of somatic cell nuclear transfer (cloning) technology.

We aim to enhance efficiency, evaluate heterogeneity of indels, develop protocols for allele introgression and broaden the utility of this method to small ruminants. We will address editing frequency (indel production by non-homologous end joining) relative to dose of editor; compare three different DNA editors - ZFN, TALEN and CRISPR/Cas9; and establish genome editing in sheep to demonstrate utility of this technology in ruminants.

In addition, we will evaluate site of delivery by comparing cytoplasmic and pronuclear delivery strategies. Current successes utilised cytoplasmic delivery of RNAs encoding the editors. Delivery to the zygote pronucleus is essential if efficient gene introgression is to be achieved using this technology. Livestock embryos are opaque, due to high lipid content. We will evaluate two methods to deliver genome editors to the pronucleus; the simple centrifugation of the zygote against the recently developed Intracellular Electroporetic Nanoinjection (IEN) method which involves electrical charge induced localised electroporation. We will compare double-stranded DNA, single-stranded DNA and oligo format DNA-templates, and different editor:DNA-template ratios will be tested.

As a further strategy, we will attempt to vectorise the DNA editor into non-integrating lentivirus vectors for delivery to the perivitelline space of the zygote. In this configuration we will compare nuclease versus nickase encoded activity, and the ability to include both the editor and the template for homology dependent recombination in the same vector.

Throughout all studies the extent of indel heterogeneity will be evaluated from sequencing of the target locus.

Planned Impact

Who will benefit from this research?

The BBSRC supported academic community. Our proposal bridges fundamental and applied science, directly addressing the BBSRC priority of 'Technology Development for the Biosciences'. In doing so it will contribute to the 'Food Security' policy, with the real potential to positively impact on Animal Health, Livestock Production, Safe and Healthy Food; and more generally to advance 'Basic Bioscience Underpinning Health'. Benefits cross other RCs will also occur; for example, refined animal biomedical models and contribution to the public debate on biotechnology.

Non-academic beneficiaries of this research include livestock breeding companies, livestock producers and ultimately the entire chain of users of animal products, including meat packers, processors, retailers and consumers. There are also benefits to the biotechnology sector, specifically companies involved in gene transfer applications.

How will they benefit from this research?

The research outputs will inform future breeding programmes in the livestock sector, enabling production of novel livestock breeds with improved performance and reduce environmental footprint. For example, in contrast to drug intervention or vaccination, genetic solutions to breeding limitations underpin sustainable improvements in production and it is now timely to explore the opportunities for engineering livestock for enhanced resilience to pathogen challenge. In addition, this type of application will enable a better understanding of the interaction between host and pathogen.

Public acceptance of genetically modified animals remains uncertain, especially in Europe. However, the development of non-transgenic livestock using genome editing technology, which introduces no exogenous DNA, has the potential to re-shape the debate. The output from this project will contribute to this dialogue.

What will be done to ensure that the benefits from this research arise?

Scientific community: Data will be made available through multiple web sites and the mainstream scientific literature. We will also present the results and publicise the resources at key scientific meetings (e.g. ISTT), seeking to engage a wide spectrum of researchers from the fields of genetics and genomics, agriculture, immunology, biomedical and veterinary pathology, and the social sciences to emphasise the cross-disciplinary of the research.

Industry: We will engage with livestock, animal health and biomedical industries through the University's commercialisation arm Edinburgh Research and Innovation. Roslin has 4 permanent business development staff who host multiple events each year, specifically showcasing the institute's work to industry partners.

Public: We will provide information about our research through our web sites (with project-specific information), talks and discussion groups and direct interaction with the media. The Roslin Institute encourages clear and open communication and has a policy of promoting Public Engagement by means of interaction with the media, presentations, publications, exhibitions and schools activities. Roslin provides support for staff and students wishing to undertake such activities. The Roslin Institute's Scientific Communications Officer in association with the Universities PR Officer oversees both internal and external communication of the research performed at the institute.

Track record: We have an excellent track record for translating the outcomes of our research and Roslin was considered an exemplar of good practice in KEC evaluation during the most recent ISPG review. We hold regular Industry Open Days, and have more than 40 existing industry partnerships. Indeed, the participation of Genus, the leading global supplier of genetically improved germplasm to the pig and cattle industries, as a project partner will greatly facilitate the realisation of the project impact in the livestock breeding sector.

Publications

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Proudfoot C (2019) Genome editing for disease resistance in pigs and chickens. in Animal frontiers : the review magazine of animal agriculture

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He Z (2015) Highly efficient targeted chromosome deletions using CRISPR/Cas9. in Biotechnology and bioengineering

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Whitelaw B (2019) We have entered the era of genome-edited farmed animals. in Emerging topics in life sciences

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LILLICO S (2020) Livestock breeding for the 21st century: the promise of the editing revolution in Frontiers of Agricultural Science and Engineering

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Hickey JM (2016) Promotion of alleles by genome editing in livestock breeding programmes. in Journal of animal breeding and genetics = Zeitschrift fur Tierzuchtung und Zuchtungsbiologie

 
Description We have demonstrated the first production of genome edited pigs, sheep and cattle by embryo injection. If GE animal are to be used in our food chain in the future then the techniques we have developed will underpin this application.
Exploitation Route This technology paves the way for future application of GE livestock on our food chain.
Sectors Agriculture, Food and Drink

URL http://www.roslin.ed.ac.uk/news/2016/02/22/pigs-genetic-code-altered-in-bid-to-tackle-deadly-virus/
 
Description We have demonstrated the use of genome editing tools for the generation of genetically engineered pigs, sheep and cattle. This as underpinned further funding from BBSRC, InnovateUK and the commercial sector. This has also generated considerable media attention.
First Year Of Impact 2015
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Description A strategic approach to identifying and combating porcine reproductive and respiratory syndrome virus outbreaks and other porcine viral diseases
Amount £787,016 (GBP)
Funding ID BB/R013187/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 03/2021
 
Description AgriTech Catayst
Amount £440,155 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 03/2015 
End 09/2016
 
Description Crop and Livestock Disease Challenge
Amount £470,000 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 05/2014 
End 05/2018
 
Description General Scheme - Bruce Whitelaw - Editing RELA S531P
Amount £30,000 (GBP)
Organisation Genus plc 
Sector Private
Country United Kingdom
Start 03/2017 
End 12/2019
 
Description General Scheme - Christine Tait-Burkard - PRRSV study at Moredun to evaluate CD163 edited pigs
Amount £48,812 (GBP)
Organisation Genus plc 
Sector Private
Country United Kingdom
Start 03/2017 
End 09/2017
 
Description Research Grant - Christine Tait-Burkard - Nanobodies as antiviral PRRSV agents
Amount £59,802 (GBP)
Organisation Eco Animal Health Ltd 
Sector Private
Country United Kingdom
Start 03/2018 
End 03/2019
 
Description Research Grant - Simon Lillico - Application of genome editors for improved bioreactor productions - goats
Amount £34,929 (GBP)
Organisation BioSourcing SA 
Sector Private
Country Belgium
Start 03/2018 
End 12/2018
 
Description Research Grant Simon Lillico - Application of genome editors for improved bioreactor production
Amount £31,675 (GBP)
Organisation BioSourcing SA 
Sector Private
Country Belgium
Start 05/2017 
End 05/2018
 
Description Responsive Mode - Christine Tait-Burkard - Understanding the CD163 - PRRS virus interaction to improve genetic engineering for resistance
Amount £674,353 (GBP)
Funding ID BB/R004463/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2017 
End 11/2020
 
Description Pig breeding with Genus 
Organisation Genus plc
Country United Kingdom 
Sector Private 
PI Contribution Developed and demonstrated gene editing in pig zygotes.
Collaborator Contribution Supply of animal resources and funds.
Impact Several publications, presentations at scientific meetings, media opportunities and joint research grants.
Start Year 2010
 
Description "Curious Minds" lecture series for the Perthshire Society of Natural Science. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Participation in the "Curious Minds" lecture series for the Perthshire Society of Natural Science. The Society runs this public lecture series in collaboration with Culture Perth and Kinross.
Year(s) Of Engagement Activity 2019
 
Description 9th International Sheep Veterinary Congress, Harrogate 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Presentation for 300 scientists on the benefits of animal biotechnology to the sheep industry
Year(s) Of Engagement Activity 2017
 
Description Academic Wine and Cheese Lecture, University of Edinburgh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Presented to University students on GM technology and implications for research
Year(s) Of Engagement Activity 2017
 
Description Animal Biotechnology Workshop, Charlottesville 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Other audiences denotes scientists. Attended an international workshop on animal biotechnology regulations where plans for international harmonisation of regulations was proposed.
Year(s) Of Engagement Activity 2017
 
Description Article in the Times: "Help on the Way for Squirrel Nutkin as Scientists gun for Grey's Anatomy 
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 Media (as a channel to the public)
Results and Impact Entry in the Times Newspaper, with research contribution for the Roslin Institute.

Grey squirrels could be wiped out in the UK by 2050 under plans to protect trees and the red squirrel population by editing the genes of the invasive species to stop the production of females.
Year(s) Of Engagement Activity 2021
 
Description BBC Country File interview 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact TV interview on genetically engineered pigs on mainstream BBC Country File programme
Year(s) Of Engagement Activity 2013
 
Description BBC Radio 4 Interview 
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 Interviewed by BBC radio on topic of genetic engineering of animals.
Year(s) Of Engagement Activity 2017
 
Description BBSRC Conclave, New Delhi 
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 Other audiences denotes scientists. UK scientific delegation to India to identify roadmap for "smart farms"
Year(s) Of Engagement Activity 2017
 
Description Centre for Tropical Livestock and Genetic Health Visit, Gates Foundation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Supporters
Results and Impact Invited to Bill Gates visit to Easter Bush Campus
Year(s) Of Engagement Activity 2017
 
Description Chair of Session 3A: Large Animal Transgenesis, TT2020 Conference, Israel 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Chair of the above online conference which sparked questions and discussion. Chance also to join in other sessions online. Most of the audience were fellow scientists
Year(s) Of Engagement Activity 2020
 
Description Collaborators' meeting, India 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Indian partnerships in industry and science meeting
Year(s) Of Engagement Activity 2019
 
Description Continuing Professional Development Talk on Animal Genetics, Glasgow 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Continuing Professional Development Talk on Animal Genetics, Glasgow to scientists and post graduate students.
Year(s) Of Engagement Activity 2019
 
Description DBT Indian visit to Roslin 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Delegated visit to discuss joint development of research programmes in livestock sector for enhancing health and productivity of farm animals.
Year(s) Of Engagement Activity 2020
 
Description Department of International Development Scientific Meeting, Nairobi 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact International event involving 100 stakeholders of the DFID and the Bill and Melinda Gates Foundation to take forward the Centre for Tropical Livestock and Genetic Health initiative
Year(s) Of Engagement Activity 2017
 
Description EuroScience Open Forum Meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Genome editing involves altering some of the individual letters that make up an organism's genetic code at precise points. The technology can be used to introduce beneficial characteristics into plants and animals, such as resistance to a specific disease or improved adaptation to different environments. The changes introduced are the same as those that could occur spontaneously in nature. In this session, we will showcase how genome editing is being researched in institutions across Europe for treatment or prevention of animal diseases. The goal of the research presented here is to improve the health and welfare of farmed animals around the world, and to improve the security of food supplies in low and middle-income countries. We will start by introducing the technology and how it works, and explore how it is being used to manage a range of diseases in salmon, pigs, and cattle. A representative from a company specialised in providing genetic solutions to the farm industry will speak about how the sector is looking at the problem. Presentations will be followed by a panel discussion and Q&A session in which the audience will be asked to consider the perspective of different groups (e.g. farmers, consumers, policy makers, researchers, low and middle income countries) and will have the opportunity to put questions to the speakers.
Year(s) Of Engagement Activity 2020
 
Description Food Evolution Film 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Participated in academic film on food biotechnology
Year(s) Of Engagement Activity 2017
 
Description Garth Vet Group, National Association, Yorkshire 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Talk given to scientists, vets and others at the Garth Vet Group, Yorkshire on animal biotechnology
Year(s) Of Engagement Activity 2019
 
Description Gene Editing in Livestock, Washington DC 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Attendance and participation in the above event talk on Genome Editing Livestock
Year(s) Of Engagement Activity 2019
 
Description Hendrix Genetcic workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Closed industry workshop on application of genetic engineering technology
Year(s) Of Engagement Activity 2016
 
Description Hendrix Genetics Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Invited participant at a closed industry meeting with Hendrix Genetics
Year(s) Of Engagement Activity 2016
 
Description International Society for Animal Genetics Conference, Lleida, Spain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Other audiences denotes scientists. Scientific symposium of 400 academic and students
Year(s) Of Engagement Activity 2019
 
Description International Sympoisum on Animal Geneticw, Dublin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Other audiences denotes scientists. Presented at scientific symposium to 400 students and students.
Year(s) Of Engagement Activity 2017
 
Description Interview with CB Films Research 
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 Discussion on how soon could we expect GM on our supermarket shelves if the government were happy to proceed with legislation now.


Discussion on resistant pigs at Roslin
Year(s) Of Engagement Activity 2020
 
Description MSc Lecture on Genome Modification of Livestock 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact MSc lecture fo 100 students on Genome Modificatio of Livestock
Year(s) Of Engagement Activity 2019
 
Description Midlorthian Science Festival Debate 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Constructive and lengthy debate

Invited back to next festival
Year(s) Of Engagement Activity 2014
 
Description National Academy of Science Colloquium Workshop, LA 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact National Academy of Scinces Colloquium Life 2.0, The Promise and Challenge of a CRISPR Path to a Sustainable Plant, Attended conference and gave talk on genome editing which I agreed could be made public
Year(s) Of Engagement Activity 2019
 
Description Nuffield Council Meeting on Bioethics 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Member of the Nuffield Council of Bioethics. Acting in an advisory capacity
Year(s) Of Engagement Activity 2019
 
Description Plant and Animal Genomics Workshop, USA 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Workshop Lead in the above workshop/conference, leading on Animal Genetics.
Year(s) Of Engagement Activity 2020
 
Description Progressional Education Trust Workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Invited to give a presentation and take part in a debate to the public on genetic engineering. PET advances public understanding of science, law and ethics in the fields of human genetics, assisted reproduction, embryology and stem cell research. Its vision is to improve the choices for people affected by infertility or genetic conditions. Its mission is to educate and debate the responsible application of reproductive and genetic science.
Year(s) Of Engagement Activity 2016
 
Description Royal Dick School of Veterinary Studies and Roslin Insitute Away Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Colleagues strategic away day to take forward and enhance the working of the Campus
Year(s) Of Engagement Activity 2020
 
Description Royal Society Genome Editing Public Consultation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Participated in Royal Society consultation of genome editing technology
Year(s) Of Engagement Activity 2017
 
Description Scientific Conference, Utrecht 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Scientific event to identify with other scientists a road map for animal biotechnology in Europe
Year(s) Of Engagement Activity 2017
 
Description Transgenic Animal Research Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Attendance at above conference as an invited speaker on Genome Modification of Livestock.
Year(s) Of Engagement Activity 2019
 
Description Transgenic Animal Research Conference, Tahoe, US 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Other audiences denotes scientist. Presented at scientific symposium for 100 students and scientists
Year(s) Of Engagement Activity 2017
 
Description U3A Talk - Rewriting Livestock, Biggar Library 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Invited talk to general public on genome editing of livestock
Year(s) Of Engagement Activity 2019
 
Description University Delegation to Bangalore India 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact University delegation to visit scientists in India to establish collaborative activities
Year(s) Of Engagement Activity 2017
 
Description University of Edinburgh filming 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Interview with University on the legacy of Dolly the Sheep
Year(s) Of Engagement Activity 2020
 
Description University of Kent, invited speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited speaker on animal biotechnology
Year(s) Of Engagement Activity 2019
 
Description named biotechnology speaker on US Ste |Department Economic Bureau Outreach Fund 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact International workshop top engage stakeholders in genetic engineering technology and applications
Year(s) Of Engagement Activity 2015