Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and a-linolenic acid phenotype in soybean

Authors: DO, PHAT - University Of Missouri; NGUYEN, CUONG - University Of Missouri; BUI, HIEN - University Of Missouri; TRAN, LY - University Of Missouri; STACEY, GARY - University Of Missouri; Gillman, Jason; ZHANG, ZHANYUAN - University Of Missouri; STACEY, MINVILUZ - University Of Missouri

Submitted to: BMC Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/25/2019
Publication Date: 7/15/2019
Publication URL: https://handle.nal.usda.gov/10113/6540733
Citation: Do, P.T., Nguyen, C.X., Bui, H.T., Tran, L.T., Stacey, G., Gillman, J.D., Zhang, Z.J., Stacey, M.G. 2019. Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and a-linolenic acid phenotype in soybean. Biomed Central (BMC) Plant Biology. 19:311. https://doi.org/10.1186/s12870-019-1906-8.
DOI: https://doi.org/10.1186/s12870-019-1906-8

Interpretive Summary: Gene editing is a powerful new molecular genetic technique capable of targeting specific genes for alteration or removal. In contrast to other methods, gene editing transgenes can subsequently be removed, leaving only small directed genetic changes behind. We tested this method by specifically disrupting two soybean seed oil biosynthetic genes; mutations for these genes have been shown to yield a more oxidatively stable and functional oil. We demonstrate that gene editing was remarkably effective in soybean; plants were readily produced which had value-added soybean oil and the trait was stable over multiple generations even without the presence of transgenic DNA. Taken together, these results demonstrate that gene editing in soybean is a remarkably useful tool either for basic research into gene function or for improving the value of the soybean crop.

Technical Abstract: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2),therefore, regulating the content of monounsaturated fats in soybean seeds. Results We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, homozygous mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation. Conclusions Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.