A rapid and efficient method to obtain germline gene editing in plants

Breeding new improved crops generally involves identifying a plant gene that brings an improved characteristic, for example increased yield or disease resistance, and then putting that improved gene into the crop variety that is grown commercially (often replacing the existing gene that was there). The new gene editing techniques that have recently been developed (and have made news headlines) now enable researchers to change the sequence of an existing gene in a plant in a very specific way so that it confers the improved characteristic without the need to transfer foreign genes into the crop plant.

Most commonly such gene editing approaches in plants involve genetic transformation and as a result those plants are classed as GMOs. This project will develop an alternative approach that will enable precise gene editing of plants for crop improvement without the need to make transgenic plants. As with gene therapy, which is used in medicine for treatment of some diseases, we will use a virus (in this case a plant virus) as a vector to get the components needed for gene editing into the plant cell. Plant viruses have long been used to express foreign genes in plants, for example for the production of vaccines. The problem is that plants have a mechanism to exclude viruses from their meristems which are the parts of the plant that go on to make flowers and seed. This is problematic for gene editing as it prevents the new improved version of the gene from being inherited in the next generation.

The novelty of this research project is that we will create a virus vector that is able to get into the meristems of plants and so the gene editing will be able to occur in the meristem. This means that seed can be harvested that contains the improved version of the gene. In this way scientists and crop breeders will be able to create a large number of gene edited plants without having to make transgenic plants.

Another advantage of being able to use this new virus vector for gene editing is that it is able to infect more than 400 species, an order of magnitude more than can be currently genetically transformed, and so it will greatly expand the number of crops that can be improved by gene editing, including many crops that are grown in developing countries.

We will use our Tobacco Rattle Virus (TRV) expression system to deliver CRISPR/Cas9 components into plant meristematic cells to obtain heritable gene edits. Using TRV as a vector means that there will be no integration of heterologous DNA into the plant genome. As TRV has a large host range this approach will enable gene editing of 400 plant species, many of which are recalcitrant to transformation. Our novel method of producing non-chimeric edited lines is transformative as it is quicker and easier, and will greatly expand the range of agricultural crops in which gene editing methods can be applied.

Virus mediated expression of transgenes in plants is well-established, and has been used to express CRISPR/Cas9 to edit genes in infected leaves, however plant meristems exclude entry of most viruses, preventing inheritance of any gene edited changes. This project will overcome this problem by using a novel RNA movement sequence that enables viral RNA to infect meristems, enabling gene editing of target genes to occur there, and thus these edits to be inherited in the seed.

We will analyse of levels of expression of the CRISPR/Cas9 genes in the TRV-infected and systemic tobacco leaves, as well as determining whether gene editing of target genes has occurred in those leaves and in the seed of infected plants. Once we have demonstrated successful inheritance of gene edited mutations, we will perform deletion analysis of the movement sequence to identify the smallest sequence required for RNA entry into the meristem.

We will test the hypothesis that if the virus is able to enter the vegetative plant meristem to cause gene editing before flower induction, there is a greater probability that the virus will be lost from the meristematic cells before they form the flower and seed. By varying the timing and tissues used for infection, it might be possible to increase the likelihood of obtaining virus-free gene edited seed in the first generation.

The outcomes of this research will be a great step forward for both industry biotechnologists and commercial plant breeders, with consequent benefits to consumers and the UK economy. The impact will be widespread in global crop improvement programmes, with potentially huge longer term benefits for developing countries.

Scientists working in Biotech companies to manipulate the function of specific genes in plants will greatly benefit from this new approach that will not only make it quicker and easier to generate a large number of new gene-edited plants, but will also make it possible to do gene-editing in a lot more plant species and varieties than is currently possible. This will have a marked impact on commercial research into specific economically important traits such as drought tolerance or disease resistance.

Plant breeders who are trying to breed new traits, such as disease resistance or drought tolerance into their elite hybrid varieties will be able to do so much quicker and easier than currently possible. This in turn will mean that new improved crop varieties will become available to the consumer much more rapidly (in 4-5 years rather than the 10-15 years it currently takes to breed a new variety). In the longer term, farmers will benefit through access to crop varieties with improved crop protection and sustainability characteristics, and this will help to maintain a continued food supply in times of changing regional climates and a rising global population.

The UK economy will benefit from the advanced breeding techniques that will be developed for UK plant breeders that will enable them to maintain, or even improve, their market position.

Developing countries will benefit economically from being able to apply gene-editing technologies to crop species and varieties that are economically important in their countries, such as in specifically-adapted varieties and/or orphan crops. Such crops/varieties are often over-looked in academic plant research as they are difficult to transform, but our research will remove that barrier to progress. During this project we will invite and host visiting researchers from developing countries to come to our lab and learn about our virus-mediated gene editing approach so that they can transfer this knowledge to laboratories in their own countries. This is something our group has a lot of experience of with students and visiting researchers coming for many years on Erasmus programmes or as part of a 5 year International Partnering Award with researchers in Hangzhou, China.