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

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 -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, null alleles, due to growth defects under stress conditions, are impractical for agronomical purposes. Here, we aimed to attenuate FAD2 activity while avoiding adverse growth effects by introducing amino-acid substitutions using CRISPR base editors. In , we applied the adenine base editor (ABE) and cytosine base editor (CBE) to induce semi-random base substitutions within several selected coding regions. Isolation of base-edited 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, 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 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.