Single and multiple gene knockouts by CRISPR–Cas9 in maize
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Key message The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts. CRISPR-Cas9 technology is a simple and efficient tool for targeted mutagenesis of the genome. It has been implemented in many plant species, including crops such as maize. Here we report single- and multiple-gene mutagenesis via stably transformed maize plants. Two different CRISPR-Cas9 vectors were used allowing the expression of multiple guide RNAs and different strategies to knockout either independent or paralogous genes. A total of 12 plasmids, representing 28 different single guide RNAs (sgRNAs), were generated to target 20 genes. For 18 of these genes, at least one mutant allele was obtained, while two genes were recalcitrant to sequence editing. 19% (16/83) of mutant plants showed biallelic mutations. Small insertions or deletions of less than ten nucleotides were most frequently observed, regardless of whether the gene was targeted by one or more sgRNAs. Deletions of defined regions located between the target sites of two guide RNAs were also reported although the exact deletion size was variable. Double and triple mutants were created in a single step, which is especially valuable for functional analysis of genes with strong genetic linkage. Off-target effects were theoretically limited due to rigorous sgRNA design and random experimental checks at three potential off-target sites did not reveal any editing. Sanger chromatograms allowed to unambiguously class the primary transformants; the majority (85%) were fully edited plants transmitting systematically all detected mutations to the next generation, generally following Mendelian segregation.
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- Authors
- Doll, N. M., Gilles, L. M., Gérentes, M.-F., Richard, C., Just, J., Fierlej, Y., Borrelli, V. M. G., Gendrot, G., Ingram, G. C., Rogowsky, P. M., & Widiez, T.
- Affiliations
- Lyon 1 Université (FR)
- Università Cattolica del Sacro Cuore (IT)
- Corresponding author
- Widiez, Thomas
- ORCID
- Venue
- Plant Cell Reports
- Published
- 2019-01-25
- Volume
- 38
- Issue
- 4
- Pages
- 487-501
- ISSN
- 0721-7714 (ISSN-L)
- 1432-203X
- OpenAlex
- W2913378721
- Cited by
- 71
- References
- 66
- Citations by year
- 2026: 4 · 2025: 7 · 2024: 10 · 2023: 9 · 2022: 15 · 2021: 11 · 2020: 12 · 2019: 3
- FWCI
- 3.40
- Type
- journal article
- Language
- en
- Open access
- green
- hal.science
- Licence
- other-oa
- www.springer.com/tdm (tdm)
- Locations
- Plant Cell Reports (publishedVersion)
- PubMed (publishedVersion)
- HAL (submittedVersion, other-oa)
- Funders
- Doctoral School on the Agro-Food System
- Association Nationale de la Recherche et de la Technologie (2018/0480, 2015/0777)
- Agence Nationale de la Recherche (ANR-11-BTBR-0001_GENIUS)
- Emergence de Jeune Equipe INRA (SeedCom)
- Ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation
- 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
- CRISPR/Cas9, maize, targeted mutagenesis, single-gene knockout, single guide RNA, paralogs, biallelic variants, off-target effects, Mendelian segregation, transgenic plants, genome editing
- MeSH terms
- Gene Editing, Zea mays, Mutagenesis, Genome, Plant, Gene Knockout Techniques, CRISPR-Cas Systems
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