Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease
Open accessAbstract
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 T0 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 T2 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.
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Numerous proof-of-concept experiments have been conducted using the CRISPR/Cas9 gene-editing platform.
“There have been numerous proof-of-concept experiments conducted using the CRISPR/Cas9 gene-editing platform (Lawrenson et al., 2015), but few developments have reached commercialization (Nonaka et al., 2017).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Lawrenson et al., 2015
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“There have been numerous proof-of-concept experi‐ ments conducted using the CRISPR/Cas9 gene-editing platform (Lawrenson et al., 2015), but few devel‐ opments have reached commercialization (Nonaka et al., 2017).”
Self-citation
- Authors
- Lawrenson, T., Shorinola, O., Stacey, N., Li, C., Østergaard, L., Patron, N., Uauy, C., & Harwood, W.
- Affiliations
- John Innes Centre (GB)
- Murdoch University (AU)
- Norwich Research Park (GB)
- Corresponding author
- Harwood, Wendy
- Venue
- Genome Biology
- Published
- 2015-11-30
- Volume
- 16
- Issue
- 1
- Pages
- 258-258
- ISSN
- 1465-6906 (ISSN-L)
- 1474-760X
- OpenAlex
- W2176470754
- Cited by
- 551
- References
- 49
- Citations by year
- 2026: 19 · 2025: 40 · 2024: 48 · 2023: 44 · 2022: 54 · 2021: 63 · 2020: 75 · 2019: 67 · 2018: 56 · 2017: 46 · 2016: 36 · 2015: 2
- FWCI
- 20.27
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Genome biology (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Murdoch University Research Portal (submittedVersion)
- University of Birmingham Research Portal (University of Birmingham) (submittedVersion, other-oa)
- Funders
- Biotechnology and Biological Sciences Research Council (BB/J004553/1)
- John Innes Centre Innovation fund
- John Innes Foundation
- Gatsby Charitable Foundation
- BBSRC and Engineering and Physics Research Council (EPSRC) Synthetic Biology Research Centre (BB/L014130/1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Genetic and Mutation Studies
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Genetically Modified Organisms Research
- Keywords
- genome editing, barley, Brassica oleracea, inherited mutations, off-target effects, single guide RNA, multicopy gene families, GA4, dwarf phenotype, transgene-free plants, gene function characterization, crop improvement
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Hordeum, Brassica, Mutation, Phenotype, RNA Editing, RNA, Guide, Kinetoplastida, Gene Targeting, Genome, Plant, CRISPR-Cas Systems
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