Increasing Monounsaturated Fatty Acid Contents in Hexaploid Camelina sativa Seed Oil by FAD2 Gene Knockout Using CRISPR-Cas9

Abstract

Seed oils are used as edible oils and increasingly also for industrial applications. Although high-oleic seed oil is preferred for industrial use, most seed oil is high in polyunsaturated fatty acids (PUFAs) and low in monounsaturated fatty acids (MUFAs) such as oleic acid. Oil from Camelina, an emerging oilseed crop with a high seed oil content and resistance to environmental stress, contains 60% PUFAs and 30% MUFAs. Hexaploid Camelina carries three homoeologs of FAD2, encoding fatty acid desaturase 2 (FAD2), which is responsible for the synthesis of linoleic acid from oleic acid. In this study, to increase the MUFA contents of Camelina seed oil, we generated CsFAD2 knockout plants via CRISPR-Cas9-mediated gene editing using the pRedU6fad2EcCas9 vector containing DsRed as a selection marker, the U6 promoter to drive a single guide RNA (sgRNA) covering the common region of the three CsFAD2 homoeologs, and an egg-cell-specific promoter to drive Cas9 expression. We analyzed CsFAD2 homoeolog-specific sequences by PCR using genomic DNA from transformed Camelina leaves. Knockout of all three pairs of FAD2 homoeologs led to a stunted bushy phenotype, but greatly enhanced MUFA levels (by 80%) in seeds. However, transformants with two pairs of CsFAD2 homoeologs knocked out but the other pair wild-type heterozygous showed normal growth and a seed MUFAs production increased up to 60%. These results provide a basis for the metabolic engineering of genes that affect growth in polyploid crops through genome editing.

Towards a Cardoon (Cynara cardunculus var. altilis)-Based Biorefinery: A Case Study of Improved Cell Cultures via Genetic Modulation of the Phenylpropanoid Pathway

“…Many biotechnological approaches have been conducted to manipulate the fatty acid composition of oilseed crops, both for industrial and food uses [ 88 ]. This has been considered over the years an important goal of research, for example, for the increase of oleic acid levels; this has been attempted mainly via the alteration of genes encoding fatty acid biosynthetic enzymes [ 89 ].…”

Realizing the Potential of Camelina sativa as a Bioenergy Crop for a Changing Global Climate

“…To complement molecular breeding approaches, advances in genetic transformation and genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) are used to improve the oil contents and fatty acid profiles in oilseed crops, including C. sativa [ 316 , 317 , 318 ]. An early demonstration of this technology in C. sativa was the simultaneous mutation of the diacylglycerol acyltransferase (DGAT1) and phospholipid:diacylglycerol acyltransferase (PDAT1) genes, which resulted in decreased oil content and altered fatty acid composition [ 319 ]. CRISPR/Cas9 disruption of all three homoeologous fatty acid desaturase 2 (FAD2) genes in C. sativa resulted in a substantial increase in oleic acid content (15% to >50%) with associated decreases in linoleic (~16% to <4%) and α-linolenic acid (~35% to <10%) content in C. sativa seeds [ 16 ].…”

“…CRISPR/Cas9 disruption of all three homoeologous fatty acid desaturase 2 (FAD2) genes in C. sativa resulted in a substantial increase in oleic acid content (15% to >50%) with associated decreases in linoleic (~16% to <4%) and α-linolenic acid (~35% to <10%) content in C. sativa seeds [ 16 ]. In a related study, disruption of all three pairs of FAD2 homeologs in C. sativa resulted in an 80% enhancement in MUFA content in seeds, but this resulted in severely stunted plants [ 319 ]. However, if only two homeologous gene pairs were knocked out, up to a 60% increase in seed MUFAs was obtained and the plants showed normal phenotypes [ 319 ].…”

“…In a related study, disruption of all three pairs of FAD2 homeologs in C. sativa resulted in an 80% enhancement in MUFA content in seeds, but this resulted in severely stunted plants [ 319 ]. However, if only two homeologous gene pairs were knocked out, up to a 60% increase in seed MUFAs was obtained and the plants showed normal phenotypes [ 319 ]. This alteration in the fatty acid profile resulted in a healthier oil profile while also improving the oxidative stability of the oil, which improves its utility for biodiesel production [ 16 ].…”

Overexpression of the vacuolar sugar importer BvTST1 from sugar beet in Camelina improves seed properties and leads to altered root characteristics

“…Without detailed analyses of metabolic fluxes and enzyme properties, it is impossible to find an explanation for this phenomenon. However, we have to keep in mind that the synthesis and composition of storage lipids and their corresponding FAs in Camelina, as in other species, depends upon the concerted action of many different enzymes, like isoforms of acyltransferases or fatty‐acid desaturase isoforms (Lee et al, 2021; Marmon et al, 2017). Thus, given that increased TST1 activity leads to higher concentrations of fatty‐acid precursors in developing seeds, we assume that the individual catalytic response of each enzyme depends upon its individual substrate affinity and its specific activity in respective cells.…”