Targeted gene modification in plants. Bacterial CRISPR systems are emerging as a promising new approach to targeted gene modification in plants1. A nuclease – Cas9 – is guided by an RNA molecule that binds by base pairing to a specific sequence motif in the plant genome where it introduces double stranded breaks that lead to deletion mutation or the potential target site for gene insertion or gene replacement. This approach provides a powerful research tool and to have application in agriculture because it is more precise than existing methods for GM2. The project will develop a refinement to CRISPR systems so that the guide RNA is delivered using a virus vector3 into lines expressing the Cas9 nuclease from a transgene. The aim is to create systems in which targeted DNA modification without the need for transgenic expression of the guide RNA as in current methods. Plants will be infected with virus vectors producing the guide RNA so that the targeted modification is established in cells of the apical meristem or floral primordium. The modification will be passed into gametes and ultimately the progeny of these plants will inherit the targeted modification without the virus or the Cas9 transgene. As a first step the student will characterize transgenic Arabidopsis lines producing Cas9 to establish that this nuclease is active with conventionally delivered guide RNAs. They will become familiar with the field of plant virology and they will design and construct virus vectors for the expression of nuclear targeted guide RNAs. Having refined and optimized the system using Arabidopsis as a model, the second stage will be to transfer the system to a crop plant and to carry out a demonstration project for eventual release into the field. This later stage of the project will provide an introduction to regulatory aspects of biotechnology and crops.
- Belhaj, K., Chaparro-Garcia, A., Kamoun, S. & Nekrasov, V. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9, 39 (2013).
- Hartung, F. & Schiemann, J. Precise plant breeding using new genome editing techniques: Opportunities, safety and regulation in the EU. Plant J. n/a–n/a (2013). doi:10.1111/tpj.12413.
- Ratcliff, F., Montserrat Martin-Hernandez, A. M. & Baulcombe, D. C. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25, 237–245 (2001).