CRISPR-Cas9 mediated targeted disruption of FAD2–2 microsomal omega-6 desaturase in soybean (Glycine max.L)

Abstract

BackgroundRecent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis.Results Here we demonstrated an efficient sequence specific integration/mutation of FAD2–2 gene in soybean using CRISPR-Cas9 nuclease system. A single guided RNA sequence was designed with the help of a number of bioinformatics tools aimed to target distinct sites of FAD2–2 loci in soybean. The binary vector (pCas9-AtU6-sgRNA) has been successfully transformed into soybean cotyledon using Agrobacterium tumafacien. Taken together our findings complies soybean transgenic mutants subjected to targeted mutation were surprisingly detected in our target gene. Furthermore, the detection of Cas9 gene, BAR gene, and NOS terminator were carried out respectively. Southern blot analysis confirmed the stable transformation of Cas9 gene into soybean. Real time expression with qRT-PCR and Sanger sequencing analysis confirmed the efficient CRISPR-Cas9/sgRNA induced mutation within the target sequence of FAD2–2 loci. The integration of FAD2–2 target region in the form of substitution, deletions and insertions were achieved with notably high frequency and rare off-target mutagenesis.ConclusionHigh frequent mutation efficiency was recorded as 21% out of all transgenic soybean plants subjected to targeted mutagenesis. Furthermore, Near-infrared spectroscopy (NIR) indicates the entire fatty acid profiling obtained from the mutants seeds of soybean. A considerable modulation in oleic acid content up to (65.58%) whereas the least level of linoleic acid is (16.08%) were recorded. Based on these finding CRISPR-Cas9 system can possibly sum up recent development and future challenges in producing agronomically important crops.Electronic supplementary materialThe online version of this article (10.1186/s12896-019-0501-2) contains supplementary material, which is available to authorized users.

Plant Unsaturated Fatty Acids: Biosynthesis and Regulation

“…For instance, transgenesis of FAD3 or DREB2C resulted in elevated 18:3 content and ameliorated multistress resistance (Shi et al, 2018;. Interestingly, to achieve high 18:1 level that is good for oil stability and human health, FAD2 has become a hotspot for targeted disruption by using CRISPR/Cas9, the powerful genome editing tool (Abe et al, 2018;Okuzaki et al, 2018;al Amin et al, 2019;Do et al, 2019). Nevertheless, crop improvement would be facilitated if the culture conditions involving light, temperature, fertilizers, and exogenous phytohormones are optimized for UFA synthesis.…”

Increased Production of α-Linolenic Acid in Soybean Seeds by Overexpression of Lesquerella FAD3-1

“…Therefore, soybeans containing low amounts of polyunsaturated fatty acids, LA and ALA, which are easily oxidized, and high oleic acid content, which is stable to oxidation at high temperatures, have been developed using genetic engineering or mutant breeding methods (Buhr et al, 2002;Graef et al, 2009;Pham et al, 2010;Pham et al, 2012). The genome editing method with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 was recently used to knockout the soybean FAD2-2 gene to achieve better production of oleic acid (Al Amin et al, 2019). However, soybean is used as a health functional food in the form of soymilk in Korea and East Asia.…”

“…Increasing the oil and protein content of soybean seed has been a task for breeders to meet the demand of the rapidly growing human population and the rising concern of food shortage (El-Hamidi and Zaher, 2018). Both quantity and quality are important factors to improve soybean oil production (Al Amin et al, 2019;Kanai et al, 2019). The fatty acids in soybean seed oil include palmitic acid (11%), stearic acid (4%), oleic acid (23%), linoleic acid (LA) (54%), and a-linolenic acid (ALA) (8%).…”

“…At present, soybean oil is mainly used for frying; hence, molecular breeding is being utilized to reduce the production of trans fat from polyunsaturated fatty acids at high temperatures. The main goal of breeding was to increase the content of oleic acid and reduce the LA and ALA levels (Flores et al, 2008;Pham et al, 2012;Yang et al, 2018;Al Amin et al, 2019). However, some contradictory reports have mentioned the benefits of high LA and ALA to human health (Rao et al, 2008;Amjad Khan et al, 2017).…”

The Potential of Genome Editing for Improving Seed Oil Content and Fatty Acid Composition in Oilseed Crops

“…Although this approach initially yields plants bearing foreign DNA, the transgene is unlinked to the edit and can thus be segregated out (or removed via the use of associated "suicide" genes expressed in pollen or embryos) while maintaining the targeted edit (He et al, 2018;Xu et al, 2018a;Yang et al, 2017a). In oilseed species such as canola, camelina, and soybean, CRISPR/Cas-mediated editing frequencies (the proportion of transgenic plants bearing an edit) ranging from 0 to 100% have been reported in the first generation when the editing machinery was introduced as a stable transgenic cassette (e.g., Al Amin et al, 2019;Aznar-Moreno and Durrett, 2017;Braatz et al, 2017;Morineau et al, 2017;Yang et al, 2018). This high level of variation likely results from differences in the target site, nature of the sgRNA, transformation method, promoters used to drive Cas and sgRNA expression, and the background vector (Ma et al, 2015;Mikami et al, 2015;Yang et al, 2017aYang et al, , 2018.…”