Journal article
Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease
Genome Biology, Vol.16(1)
2015
Abstract
Background
The RNA-guided Cas9 system represents a flexible approach for genome editing in plants. This method can create specific mutations that knock-out or alter target gene function. It provides a valuable tool for plant research and offers opportunities for crop improvement.
Results
We investigate the use and target specificity requirements of RNA-guided Cas9 genome editing in barley (Hordeum vulgare) and Brassica oleracea by targeting multicopy genes. In barley, we target two copies of HvPM19 and observe Cas9-induced mutations in the first generation of 23 % and 10 % of the lines, respectively. In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened. In addition, a phenotypic screen identifies T 0 plants with the expected dwarf phenotype associated with knock-out of the target gene. In both barley and B. oleracea stable Cas9-induced mutations are transmitted to T 2 plants independently of the T-DNA construct. We observe off-target activity in both species, despite the presence of at least one mismatch between the single guide RNA and the non-target gene sequences. In barley, a transgene-free plant has concurrent mutations in the target and non-target copies of HvPM19.
Conclusions
We demonstrate the use of RNA-guided Cas9 to generate mutations in target genes of both barley and B. oleracea and show stable transmission of these mutations thus establishing the potential for rapid characterisation of gene function in these species. In addition, the off-target effects reported offer both potential difficulties and specific opportunities to target members of multigene families in crops.
Details
- Title
- Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease
- Authors/Creators
- T. Lawrenson (Author/Creator) - Norwich Research ParkO. Shorinola (Author/Creator) - Norwich Research ParkN. Stacey (Author/Creator) - Norwich Research ParkC. Li (Author/Creator) - Murdoch UniversityL. Østergaard (Author/Creator) - Norwich Research ParkN. Patron (Author/Creator) - Sainsbury LaboratoryC. Uauy (Author/Creator) - Norwich Research ParkW. Harwood (Author/Creator) - Norwich Research Park
- Publication Details
- Genome Biology, Vol.16(1)
- Publisher
- BioMed Central
- Identifiers
- 991005540386907891
- Copyright
- © 2015 Lawrenson et al.
- Murdoch Affiliation
- State Agricultural Biotechnology Centre
- Language
- English
- Resource Type
- Journal article
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- Collaboration types
- Domestic collaboration
- International collaboration
- Citation topics
- 1 Clinical & Life Sciences
- 1.54 Molecular & Cell Biology - Genetics
- 1.54.1401 Genome Editing
- Web Of Science research areas
- Biotechnology & Applied Microbiology
- Genetics & Heredity
- ESI research areas
- Molecular Biology & Genetics