Refining genome editing in livestock

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.

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.

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.