A CRISPR/Cas9 toolkit for multiplex genome editing in plants
Open accessAbstract
BACKGROUND: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. RESULTS: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. CONCLUSIONS: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.
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Advancement of the technique to multiplex gene editing allows the simultaneous editing of several targets in a single genome.
“Advancement of the technique to multiplex gene editing allows the simultaneous editing of several targets in a single genome (Xing et al., 2014).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Xing et al., 2014
Multiplex gene editing can solve the problem of traits controlled by a few QTL by facilitating the simultaneous editing of two or more genes, or multiple loci of the same gene.
“Multiplex gene editing can solve this problem by facilitating the simultaneous editing of two or more genes, or multiple loci of the same gene (Xing et al., 2014).”
From Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance (2023) · cites this paper as Xing et al., 2014
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- Authors
- Xing, H.-L., Dong, L., Wang, Z.-P., Zhang, H.-Y., Han, C.-Y., Liu, B., Wang, X.-C., & Chen, Q.-J.
- Affiliations
- State Key Laboratory of Plant Physiology and Biochemistry (CN)
- Corresponding author
- Chen, Qi-Jun
- Venue
- BMC Plant Biology
- Published
- 2014-11-29
- Volume
- 14
- Issue
- 1
- Pages
- 327-327
- ISSN
- 1471-2229 (ISSN-L)
- PMC
- PMC4262988
- OpenAlex
- W2165016943
- Cited by
- 1,684
- References
- 50
- Citations by year
- 2026: 162 · 2025: 214 · 2024: 214 · 2023: 197 · 2022: 211 · 2021: 174 · 2020: 159 · 2019: 113 · 2018: 89 · 2017: 64 · 2016: 56 · 2015: 31
- FWCI
- 20.62
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- BMC Plant Biology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Natural Science Foundation of China (31070329)
- National Key Research and Development Program of China (2012CB114200)
- National Transgenic Science and Technology Program (2011ZX08009)
- 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 › Biochemistry, Genetics and Molecular Biology › Biotechnology › Transgenic Plants and Applications
- Keywords
- CRISPR/Cas9, multiplex genome editing, binary vectors, transgenic plants, Arabidopsis mutants, selectable marker
- MeSH terms
- Base Sequence, Zea mays, Genetic Engineering, Genetic Vectors, Protoplasts, Sequence Alignment, Arabidopsis, Genome, Plant, Plants, Genetically Modified, Agrobacterium, CRISPR-Cas Systems
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