C-to-G Base Editing Enhances Oleic Acid Production by Generating Novel Alleles of FATTY ACID DESATURASE 2 in Plants

Abstract

The demand for vegetable oil, which is mainly used for dietary purposes and cooking, is steadily increasing worldwide. It is often desirable to reduce unsaturation levels of fatty acids in order to increase storage stability and reduce trans-fat generation during cooking. Functional disruption of FATTY ACID DESATURASE 2 (FAD2) prevents the conversion of monounsaturated oleic acid to polyunsaturated linoleic acid, thereby enhancing the production of the desirable oleic acid. However, FAD2 null alleles, due to growth defects under stress conditions, are impractical for agronomical purposes. Here, we aimed to attenuate FAD2 activity in planta while avoiding adverse growth effects by introducing amino-acid substitutions using CRISPR base editors. In Arabidopsis, we applied the adenine base editor (ABE) and cytosine base editor (CBE) to induce semi-random base substitutions within several selected FAD2 coding regions. Isolation of base-edited fad2 alleles with higher oleic acid revealed that the CBE application induced C-to-T and/or C-to-G base substitutions within the targeted sequences, resulting in an alteration of the FAD2 enzyme activities; for example, fad2-144 with multiple C-to-G base substitutions showed less growth defects but with a significant increase in oleic acids by 3-fold higher than wild type. Our “proof-of-concept” approach suggests that equivalent alleles may be generated in vegetable oil crops via precision genome editing for practical cultivation. Our targeted semi-random strategy may serve as a new complementary platform for planta engineering of useful agronomic traits.

Applications and prospects of genome editing in plant fatty acid and triacylglycerol biosynthesis

“…When FAD2 loses its activity, the PUFA content of the seed oil decreases, and the oleic acid content increases; however, it is sensitive to salt stress during seed germination and seedling growth ( Zhang et al, 2012 ). Recently, various fad2 alleles have been reported to weaken the function of FAD2 through base editing, increase oleic acid content, and confer resistance to salt stress ( Park et al, 2021 ).…”

“…In addition, the base editor can be a good strategy to change one amino acid or mutate randomly in the plant genome ( Figure 3D ). For example, with random mutation of the FAD2 gene of Arabidopsis using base editing, the activity of FAD2 was weakened, and individuals with high oleic acid and resistance to salt stress were selected ( Park et al, 2021 ).…”

“…To avoid such extreme phenotypes, it is necessary to specifically knockout only one or two FAD2 genes in camelina ( Lee et al, 2021 ). In addition, in plants with a single copy of FAD2 , it may be preferable to create a weak allele using the base editor ( Park et al, 2021 ). A potential problem is that Cas9 can bind to unintended sites causing accidental mutations, or off-target mutations.…”