Targeted gene knockouts in crops using RNA-guided Cas9 nuclease

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.

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.

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.