Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family

• Published in: International journal of molecular sciences Vol. 24; no. 5; p. 4769
• Main Authors: Zhou, Junming, Li, Zeyuan, Li, Yue, Zhao, Qiuzhu, Luan, Xinchao, Wang, Lixue, Liu, Yixuan, Liu, Huijing, Zhang, Jun, Yao, Dan
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01-03-2023; MDPI
• Subjects: Amino acids; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; Editing; editing vector; Efficiency; Enzymes; FAD2 gene; Fatty Acid Desaturases - genetics; Fatty acids; Fatty Acids - metabolism; Food supply; Gene Editing - methods; Genes; Genetic engineering; Genetic modification; Genetic transformation; Genome editing; Genomes; Glycine max - genetics; GmFAD2; Linoleic acid; Metabolism; Mutation; Oleic acid; Oleic Acid - metabolism; Plant breeding; Plants, Genetically Modified - genetics; Progeny; Proteins; Seeds; soybean; Soybeans; Transgenic plants; China; soybean; CRISPR/Cas9; fatty acids; editing vector; GmFAD2
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24054769

Δ
-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean
gene family-
,
,
,
, and
-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T
generation were obtained using
-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for
. The phenotypic analysis revealed that the oleic acid content of the progeny of
gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were
,
,
, and
. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.