TY - JOUR
T1 - Multiple sgRNAs for one-step inactivation of the duplicated acetyl-coenzyme A carboxylase 2 (ACC2) genes in Brassica napus
AU - LaManna, Lisa M.
AU - Parulekar, Mugdha S.
AU - Maliga, Pal
N1 - Publisher Copyright: © 2022 American Society of Plant Biologists. All rights reserved.
PY - 2022/5
Y1 - 2022/5
N2 - 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.
AB - 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.
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U2 - 10.1093/plphys/kiac069
DO - 10.1093/plphys/kiac069
M3 - Article
C2 - 35188200
SN - 0032-0889
VL - 189
SP - 178
EP - 187
JO - Plant physiology
JF - Plant physiology
IS - 1
ER -