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The low-input oilseed crop Camelina (Camelina sativa (L.) Crantz) is known for its high omega-3 (18:3) content, short growth season, and facile gene transformation. Camelina mostly contains unsaturated fatty acids, however its fatty acid composition needs optimization depending on the end uses, for example reduction of unsaturated fatty acid to use as biodiesels, or enhancing omega-3 fatty acid content to use as nutritional supplements. Very long chain fatty acid (VLCFAs, C20-C24), are undesirable for human consumption, and their accumulation in seed oil also needs to be diminished. VLCFAs are produced by the catalytic action of fatty acid elongase1 (FAE1), and Camelina contains three alleles of FAE1 genes (FAE1-A, FAE1-B, and FAE1-C) due to its allohexaploid nature. Recently, VLCFAs in camelina were decreased along with polyunsaturated fatty acids (PUFAs) using the RNA interference (RNAi) technology. A low VLCFA line was also isolated from ethyl methanesulfonate (EMS) induced mutants. Sequencing results indicated that FAE1-B gene was mutated and resulted in 60% reduction in VLCFAs, but other two FAE1 copies were presumably still active in the mutant. To address this multipleallele-knockout-at-once problem, here I investigated the effect of knocking out three alleles of FAE1 genes using CRISPR technology with egg cell-specific Cas9 expression. Due to the germline mutation, homozygous FAE1 knockout mutants were successfully created in a single generation. VLCFA accumulation was significantly decreased from 22% of total fatty acids in wild type to less than 2% in transgenic plants, and the C18 unsaturated fatty acids were improved since 18:1 substrates were diverted to desaturation pathway, rather than elongation. Analysis of the fatty acid composition of four transgenic generations indicated that the mutations that cause low VLCFA genotype were heritable. There was no significant difference observed in seed weight, plant height, total oil content, and seed germination in Cas9-induced mutants compared to the wild type. This study showed that polyploid Camelina can be modified rapidly and effectively through CRISPR/Cas9 to achieve desired fatty acid composition. |
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