Development of specific TALENs for precision engineering in wheat

Recent developments in genome engineering mean that researchers now have tools which can be used to modify specific sequences within any species. One of these tools, called TALENs (transcription activator-like effector nucleases) could be of significant benefit to wheat researchers as they might allow plant geneticist and plant breeders to rapidly modify a crop to have unique agronomic properties while at the same time eliminating the need for non-host DNA to remain within the crop. Such improvements might include increased disease resistance, increased yield or better bread making qualities. While TALENs could revolutionise wheat breeding the system is not yet been fully tested, this is important as wheat is a polyploid (consisting of three different sets of chromosomes) and it has a large genome (consisting of approximately 5 times more DNA that the human genome). Hence, before TALENs can be applied to wheat, or any other crop, on a large scale, they need to be tested and monitored to confirm their early promise. Here we propose to test and monitor TALENs in wheat. To carry out this testing and monitoring we propose to utilise TALENs specific of two wheat genes, furthermore we propose to test the efficiency of TALEN activity when they are placed under the control of two promoters, one that is permanently switched on and one that can be induced by an increase in temperature.

Following the introduction of the TALEN constructs into the wheat plant we will monitor their activity in terms of both their effectiveness at mutating the target genes and for NOT modifying other regions of the genome. Hence, one outcome of the project would be guidelines and protocols designed to ensure that researchers use the most effective promoters and gene sequences to increase ON target activity whilst simultaneously reducing or eliminating OFF target activity. Following successful completion of this preliminary study we will be well placed to advise government agencies on the effectiveness of TALENs and the steps that need to be put into place to ensure their safe use in future plant breeding.

Recent advances in the field of genome engineering make it possible to create site-directed modifications to the genomes of crop species. This type of genome engineering is mediated by site-directed nucleases (SDN) and includes targeted mutations, gene insertions, and gene replacements.

One of the major technical bottlenecks in using SDNs is the highly variable nature of the mutations seen in the regenerating primary transgenic plants. This variation can be both spatially (cell-to-cell) and temporally (developmental age). Although the mutagenic action of these endonucleases appears to be targeted to a specific DNA sequence, the repair mechanisms that generate the mutations is independent in every cell in the regenerating plant. Thus at the DNA level, there will be many different examples of DSB repair or gene editing in different cells at the same time in the same plant. Because the cell lineages that form male and female germ lines deviate relatively early in development, it is probable that pollen nuclei will possess different mutations compared to the egg cell nuclei. Given this unpredictable nature it is probable that many of the mutations observed in the T0 generation will be lost in the following generation.

To quantify the problems highlighted above and to test a possible solution, Rothamsted Research and Bristol University intend to work together to compare the performance of wheat transformed with TALEN constructs under the control of one of two promoters; the constitutive maize ubiquitin 1 promoter and the barley heat shock promoter (HSP). The results of this analysis could not only generate new protocols for the manipulation of the wheat genome but it would also provide valuable information of use to government agencies when formulating policy in this important area.

Who will benefit?
Academics and research scientists, wheat breeding companies and agribiotechnology companies, regulatory authorities and risk assessment bodies, policy makers.

How will they benefit?
The data and knowledge generated in this project will inform future academic research, commercial plant breeding and policy decisions regarding biotechnology risk assessment. This new technology is predicted to revolutionize functional genomics research and could generate new and valuable traits for commercial plant breeding. The commercial aspect of SDN's is relevant because there are prospects that GMO regulatory authorities will treat this type of genome editing more like conventional mutagenesis then recombinant DNA technology.
To realise the full impact of the research, once preliminary data has been generated and quality checked and once any suitable IP has been acquired, we intend to present the results at both at the 2015 Monogram Workshop and, via personal invitations, to the UK-based wheat breeding companies and Agri-Biotechnology companies, including Syngenta, Monsanto, Limagrain, KWS and RAGT. We will ensure that the presentations include detailed discussions on how the work might be taken forward as collaborative projects and how the work might be expanded to include other crops such as barley and oil seed rape.

As Rothamsted already offers a wheat transformation service, Rothamsted will explore ways to promote the use of the inducible TALEN technology among its service users, while Bristol University will focus its efforts on delivering further impact via its interests in modifying recombination rates in regions of the wheat genome current devoid of cross over activities.

Both HDJ and KJE have an excellent track record of generating impact. In all cases this has been achieved by communicating the results of their BBSRC funded research to specific stakeholders and the wider community. For instance HDJ represents the BBSRC on the Global Food Security Programme Communication Group and is a member of the BBC Rural Affairs Committee and the Monogram steering committee. As well as academic audiences, he regularly speaks to special interest groups such as bee-keepers and various farming organisations. As the holder of the Defra licence for the current GM wheat trial, he also has experience of addressing the broad scientific and societal issues of GM crops via various broadcast and print media. As part of his role in various BBSRC funded projects, KJE has continually ensured that information (sequence and genotyping data) and the associated tools and resources, has been made available in a variety of forms, to the wider community, for instance as part of his role as PI on the BBSRC funded project "Mining the allohexaploid wheat genome for useful sequence polymorphisms" he was jointly responsible (along with co-PIs from Liverpool and the JIC) with ensuring that the 5 fold sequence coverage of the wheat genome was both fully released and made available for screening (via the CerealsDB web site) before publication. The Independent newspaper called this event "the most significant breakthrough in wheat production in 10,000 years" (27th August 2010). In recognition of the impact that his work has had on wheat genetics, the Royal Agricultural Society of England awarded him their 2011 Research Medal.
In addition to delivering academic impact, both HDJ and KJE have a track record of interacting with industry, specifically wheat breeders and the agri-biotechnology sector, hence both PIs are well placed to present the work carried out to those end users who will directly benefit from the developments that the project will enable.