Multiple sgRNAs for one-step inactivation of the duplicated acetyl-coenzyme A carboxylase 2 (ACC2) genes in Brassica napus

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 tran...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 2022-05, Vol.189 (1), p.178-187
Main Authors: LaManna, Lisa M, Parulekar, Mugdha S, Maliga, Pal
Format: Article
Language:English
Subjects:
Acetyl-CoA Carboxylase - genetics
Acetyl-CoA Carboxylase - metabolism
Arabidopsis - genetics
Arabidopsis - metabolism
Brassica napus
Coenzyme A
RNA, Guide, CRISPR-Cas Systems
Spectinomycin
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Summary: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.
ISSN:0032-0889
1532-2548
DOI:10.1093/plphys/kiac069