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

Abstract

Δ12-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 FAD2 gene family—GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C—and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A 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 GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. 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.

Advances in CRISPR/Cas9-based research related to soybean [Glycine max (Linn.) Merr] molecular breeding

“…analyzed the oleic acid contents of soybean plants over-expressing Gm15G117700 and soybean plants in which the gene was edited; the oleic acid content increased in the gene-edited plants by 3.49% ( Qu et al., 2021 ). Zhou et al. (2023a) recently edited five important enzyme-encoding genes in the GmFAD2 family and analyzed the associated effects on soybean oil synthesis.…”

“…(2023a) recently edited five important enzyme-encoding genes in the GmFAD2 family and analyzed the associated effects on soybean oil synthesis. Editing GmFAD2-1A increased the oleic acid content by 91.49% ( Zhou et al., 2023a ). In another recent study, Li et al.…”

“…In the field of molecular breeding, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a commonly used third-generation gene editing technology ( Nakamori, 2023 ). Thus, many new and desirable soybean traits have been developed using gene editing technology, which is currently a hot topic in scientific research ( Osakabe and Osakabe, 2017 ; Chen et al., 2022 ; Zhou et al., 2023a ).…”