Genome editing in wheat with CRISPR/Cas9

Abstract

Genetically engineered crops have the potential to play a key role in achieving global food security and transitioning to a more sustainable agriculture. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for crop genome editing. CRISPR/Cas9 enables the targeted and precise modification of plant genomes via the creation and subsequent repair of site-specific DNA double-strand breaks (DSBs). The system consists of the Cas9 endonuclease in complex with a small guide RNA (gRNA) that is designed to target a specific site in the genome. Site-specific DSBs generated by Cas9 are repaired through non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ is error-prone and often produces small insertions or deletions (indels) that result in gene knockout. Alternatively, if an exogenous DNA donor template is delivered to the cell, then precise modifications can be made via HDR. The CRISPR/Cas9 system has been successfully applied to many model and crop plants. However, it can be difficult to achieve highly efficient and specific editing in polyploid species. Therefore, the main aim of this PhD project was to develop tools and methods for optimising the CRISPR/Cas9 for efficient and specific genome editing in hexaploid bread wheat (Triticum aestivum). To test the efficacy of the CRISPR/Cas9 system for gene knockout, three gRNAs were designed to target Ms1, a male fertility gene that has been proposed for use in hybrid seed production. CRISPR/Cas9 vectors were delivered to immature embryos via Agrobacterium-mediated stable transformation, and the regenerated T0 lines were screened for targeted indels produced via NHEJ. Only one of the three gRNAs was efficacious. Five per cent (2/40) of T0 lines carrying the active gRNA were edited and male sterile, whereas all unedited lines were fully fertile. The recessive mutations were stably transmitted to the T1, T2 and T3 generations, as was the male sterile phonotype. Given the observed variability in the efficacy of different gRNAs targeting the same gene, and given that wheat transformation and tissue culture takes months and is laborious, a method was developed for the rapid assessment of gRNA activity and specificity. Seven gRNAs were designed to target EPSPS, a gene involved in aromatic amino acid biosynthesis. CRISPR/Cas9 vectors were then transiently transformed into wheat protoplasts. Three out of the seven gRNAs induced mutations at moderate to high frequencies. gRNA specificity was correlated with the number and distribution of mismatches in the ‘seed’ region of the gRNA. One of the gRNAs was selected as potentially suitable for the development of non-transgenic herbicide resistant wheat lines.