Dataset Information

CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

ABSTRACT: The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in 'Chardonnay' suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape.

SUBMITTER: Ren C

PROVIDER: S-EPMC5006071 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

Ren Chong C Liu Xianju X Zhang Zhan Z Wang Yi Y Duan Wei W Li Shaohua S Liang Zhenchang Z

Scientific reports 20160831

The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in 'Chardonnay' suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of ...[more]

PMID: 27576893

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Project description:Sweet basil (Ocimum basilicum) is an economically important herb and its global production is threatened by basil downy mildew caused by the obligate biotrophic oomycete Peronospora belbahrii. Effective tools are required for functional understanding of its genes involved in synthesis of valuable secondary metabolites in essential oil and disease resistance, and breeding for varieties with improved traits. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene editing technology has revolutionized crop breeding and functional genomics. The applicability and efficacy of this genomic tool in the allotetraploid sweet basil were tested by editing a potential susceptibility (S) gene ObDMR1, the basil homolog of Arabidopsis DMR1 (Downy Mildew Resistant 1) whose mutations conferred nearly complete resistance against Arabidopsis downy mildew pathogen, Hyaloperonospora arabidopsidis. Two single guide RNAs targeting two different sites of the ObDMR1 coding sequence were designed. A total of 56 transgenic lines were obtained via Agrobacterium-mediated stable transformation. Mutational analysis of 54 T0 transgenic lines identified 92.6% lines carrying mutations at target 1 site, while a very low mutation frequency was detected at target 2 site. Deep sequencing of six T0 lines revealed various mutations at target 1 site, with a complete knockout of all alleles in one line. ObDMR1 homozygous mutant plants with some being transgene free were identified from T1 segregating populations. T2 homozygous mutant plants with 1-bp frameshift mutations exhibited a dwarf phenotype at young seedling stage. In summary, this study established a highly efficient CRISPR/Cas9-mediated gene editing system for targeted mutagenesis in sweet basil. This system has the capacity to generate complete knockout mutants in this allotetraploid species at the first generation of transgenic plants and transgene-free homozygous mutants in the second generation. The establishment of this system is expected to accelerate basil functional genomics and breeding.

Dataset Information

CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

Publications

CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

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