Targeted gene knockouts in crops using RNA-guided Cas9 nuclease

Lead Research Organisation: Earlham Institute
Department Name: UNLISTED

Abstract

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. 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. Over the project we will also engage in a range of activities to inform potential users of the resource and to provide information on genome editing to stakeholder groups

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