Generation of Transgene-Free Semidwarf Maize Plants by Gene Editing of Gibberellin-Oxidase20-3 Using CRISPR/Cas9

Zhang, Jiaojiao; Zhang, Xiaofeng; Chen, Rongrong; Yang, Li; Fan, Kaijian; Liu, Yan; Wang, Guoying; Ren, Zhenjing; Liu, Yun-Jun

doi:10.3389/fpls.2020.01048

Cited by 44 publications

(20 citation statements)

References 41 publications

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“…RNP delivered in plants [12] Arabidopsis thaliana, Lactuca sativa, Nicotiana attenuata, Oryza sativa Protoplasts PEG-mediated 5.7-40.0% [13] Malus domestica, Vitis vinifera Protoplasts PEG-mediated 0.1-6.9% [14] Petunia x hybrid Protoplasts PEG-mediated 2.4-21.0% [31] Triticum aestivum Protoplasts, immature embryos PEG-mediated, Biolistics 0.2-45.3% [15] Solanum tuberosum Protoplasts PEG-mediated 1.0-25.0% [32] Brassica oleracea, Brassica rapa Protoplasts PEG-mediated 0.1-24.5% [33] Oryza sativa Zygotes PEG-mediated 14.0-64.0% Zea mays Immature embryos Agrobacterium-mediated 12.0-74.0% [38] Zea mays Immature embryos Vector via Biolistics 60.0-98.0% [39] Zea mays Immature embryos Agrobacterium-mediated N.A [19] Zea mays Immature embryos Agrobacterium-mediated 5.0-100% [20] Zea mays Immature embryos Agrobacterium-mediated N.A [40] Zea mays Immature embryos Agrobacterium-mediated 90.0-100% [41] Zea mays Immature embryos Agrobacterium-mediated N.A [42] Zea mays Immature embryos Agrobacterium-mediated N.A [43] Zea mays Immature embryos Agrobacterium-mediated N.A [44] Zea mays Immature embryos Agrobacterium-mediated N.A [45] Zea mays Immature embryos Agrobacterium-mediated N.A [46] Zea mays Immature embryos Vector via biolistics N.A [47] Zea mays Immature embryos Agrobacterium-mediated 25.0-100% [48] Zea mays Immature embryos Agrobacterium-mediated N.A Present study Zea mays Protoplasts RNA via PEG-mediated 0.85-5.85% Note: N.A is 'not applicable' because transgenic plants were either selected using antibiotic marker genes or the analysis was not performed.…”

Section: Reference Plant Species Plant Materials Transfection Methods Gene-editing Efficiencymentioning

confidence: 99%

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Sant’Ana

Caprestano

Nodari

et al. 2020

Genes

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.

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Section: Reference Plant Species Plant Materials Transfection Methods Gene-editing Efficiencymentioning

confidence: 99%

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Sant’Ana

Caprestano

Nodari

et al. 2020

Genes

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“…The analysis of hormone biosynthesis, transport and signalling has achieved more promising results. Knocking out the gibberellin-oxidase20-3 gene blocked the gibberellin biosynthesis pathway and generated semi dwarf maize plants with increased lodging resistance (Zhang et al 2020b). Similarly, using CRISPR/Cas9 to target the BRACHYTIC2 (BR2) gene, which encodes an ATP binding cassette type B (ABCB) involved in auxin transport, induced a 1-bp frameshift that generated a premature stop codon in exon 5 and resulted in a semi dwarf phenotype similar to natural br2 mutations.…”

Section: Maizementioning

confidence: 99%

Genome editing in cereal crops: an overview

et al. 2021

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Genome-editing technologies offer unprecedented opportunities for crop improvement with superior precision and speed. This review presents an analysis of the current state of genome editing in the major cereal crops- rice, maize, wheat and barley. Genome editing has been used to achieve important agronomic and quality traits in cereals. These include adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Not all traits can be achieved through genome editing, and several technical and regulatory challenges need to be overcome for the technology to realize its full potential. Genome editing, however, has already revolutionized cereal crop improvement and is poised to shape future agricultural practices in conjunction with other breeding innovations.

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“…Drought stress-tolerant genome-edited maize has been developed by precise modification of the promoter region to increase the expression of the ARGOS8 gene ( Shi et al, 2017 ). Other genome-edited maize varieties currently being developed include varieties with the traits of male sterility to facilitate hybrid development ( Wan et al, 2019 ), tolerance to multiple stresses ( Zhou et al, 2016 ), and dwarfism ( Zhang et al, 2020b ). From 2018 to 2020, several companies invested in genome editing in maize for achieving drought tolerance, resistance, and increased yield and stability, in addition to several traits investigated by government or academic institutions ( USDA-APHIS, 2021 ).…”

Section: Development Of Genetically Modified and Edited Varietiesmentioning

confidence: 99%

Maize Transformation: From Plant Material to the Release of Genetically Modified and Edited Varieties

et al. 2021

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Over the past decades, advances in plant biotechnology have allowed the development of genetically modified maize varieties that have significantly impacted agricultural management and improved the grain yield worldwide. To date, genetically modified varieties represent 30% of the world’s maize cultivated area and incorporate traits such as herbicide, insect and disease resistance, abiotic stress tolerance, high yield, and improved nutritional quality. Maize transformation, which is a prerequisite for genetically modified maize development, is no longer a major bottleneck. Protocols using morphogenic regulators have evolved significantly towards increasing transformation frequency and genotype independence. Emerging technologies using either stable or transient expression and tissue culture-independent methods, such as direct genome editing using RNA-guided endonuclease system as an in vivo desired-target mutator, simultaneous double haploid production and editing/haploid-inducer-mediated genome editing, and pollen transformation, are expected to lead significant progress in maize biotechnology. This review summarises the significant advances in maize transformation protocols, technologies, and applications and discusses the current status, including a pipeline for trait development and regulatory issues related to current and future genetically modified and genetically edited maize varieties.

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Generation of Transgene-Free Semidwarf Maize Plants by Gene Editing of Gibberellin-Oxidase20-3 Using CRISPR/Cas9

Cited by 44 publications

References 41 publications

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Genome editing in cereal crops: an overview

Maize Transformation: From Plant Material to the Release of Genetically Modified and Edited Varieties

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