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• W4213309032 abstract "Efficient plastid transformation in Arabidopsis (Arabidopsis thaliana) requires genetic lines that are hypersensitive to spectinomycin due to the absence of a chloroplast acetyl-coenzyme A carboxylase (ACCase) encoded in the acetyl-coenzyme A carboxylase 2 (ACC2) nuclear gene. To obtain plastid transformation-competent oilseed rape (Brassica napus), we inactivated all nuclear encoded, chloroplast targeted ACCase copies using CRISPR-Cas9. Brassica napus (2n = 38, AACC) is a recent interspecific hybrid of Brassica rapa (2n = 20, AA) and B. oleracea (2n = 18, CC) and is expected to have at least two ACC2 copies, one from each parent. The sequenced genome has two ACC2 copies, one that is B. rapa-like and one that is B. oleracea-like. We designed single-guide RNAs (sgRNAs) that could simultaneously inactivate both nuclear ACC2 copies. We expressed Cas9 from a chimeric egg cell promoter 1.2 (EC1.2p) known to yield homozygous or biallelic mutants in Arabidopsis in the T1 generation. To maximize the probability of functionally inactivating both orthologs in a single step, each of the two vectors carried four sgRNAs. Four T0 transgenic lines were obtained by Agrobacterium tumefaciens-mediated hypocotyl transformation. Amplicon sequencing confirmed mutations in ACC2 genes in 10 T1 progeny, in seven of which no wild-type (WT) copy remained. The B. napus T2 seedlings lacking WT ACC2 gene copies exhibited a spectinomycin hypersensitive phenotype, suggesting that they will be a useful resource for chloroplast genome transformation." @default.
• W4213309032 created "2022-02-24" @default.
• W4213309032 creator A5009809779 @default.
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• W4213309032 date "2022-02-21" @default.
• W4213309032 modified "2023-09-27" @default.
• W4213309032 title "Multiple sgRNAs for one-step inactivation of the duplicated <i>acetyl-coenzyme A carboxylase 2</i> (<i>ACC2</i>) genes in <i>Brassica napus</i>" @default.
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• W4213309032 doi "https://doi.org/10.1093/plphys/kiac069" @default.
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