Page 313 - ebook

[F. Others] F-13

C-to-G Base Editing Increases Oleic Acid Production without

any growth defects

Mid-Eum Park¹, Yuli Choe², Hyun Uk Kim¹,²*

¹Department of Molecular Biology, Plant Engineering Research Institute, Seoul 05006, Korea, ²Bioindustry and

Bioresource Engineering, Plant Engineering Research Institute, Seoul 05006, Korea

The demand for vegetable oil is steadily increasing worldwide for dietary purposes and cooking. It is often desirable

to reduce unsaturation levels of fatty acids to increase storage stability and prevent vegetable oils from turning
rancid and reduce the amount of trans-fat generation during cooking. Functional disruption of FATTY ACID

DESATURASE 2 (FAD2) prevents the conversion of oleic acid (18:1) to linoleic acid (18:2), thereby enhancing the
production of the desirable oleic acid. 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

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 fewer growth defects
but with a significant increase in oleic acids by 3-fold higher than wild type. Our “proof-of-concept” approach

suggests that the equivalent alleles may be recapitulated in vegetable oil crops via precision genome editing for
practical cultivation.