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

Mid-Eum Park; Jae-Young Yun; Hyun Uk Kim

doi:10.3389/fpls.2021.748529

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

Front Plant Sci. 2021 Oct 26:12:748529. doi: 10.3389/fpls.2021.748529. eCollection 2021.

Authors

Mid-Eum Park¹, Jae-Young Yun², Hyun Uk Kim^{1

3}

Affiliations

¹ Department of Molecular Biology, Graduate School, Sejong University, Seoul, South Korea.
² Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang, South Korea.
³ Department of Bioindustry and Bioresource Engineering, Plant Engineering Research Institute, Sejong University, Seoul, South Korea.

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.

Keywords: CRISPR; FAD2; base editing; cytosine base editor; oleic acid; vegetable oil.