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Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing.

ABSTRACT: The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T₂ and T₃ generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T₃ and T₄ generation Camelina seeds was associated with a combination of germ-line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.

SUBMITTER: Jiang WZ

PROVIDER: S-EPMC5399004 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing.

Jiang Wen Zhi WZ Henry Isabelle M IM Lynagh Peter G PG Comai Luca L Cahoon Edgar B EB Weeks Donald P DP

Plant biotechnology journal 20170112 5

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were as ...[more]

PMID: 27862889

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Project description:BackgroundThe oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds.ResultsCRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids.ConclusionsThis study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.

Project description:Simple Summary Modern livestock research has focused on the evaluation of feeding strategies that led to modify the rumen microbiome to achieve optimum productivity without compromising ruminants’ physiology and health. For this reason, the supplementation of unconventional feedstuffs is extensively studied. In our study, we investigated the effect of Camelina sativa seeds supplementation on ewe’s rumen microbiota. Our results suggested that supplementing Camelina sativa seeds, especially in the highest studied level (160 g·kg−1 of concentrate), resulted in significant alterations in the relative abundance of the rumen microorganisms, with those reported in methanogens being considered the most promising. Abstract Supplementing ruminant diets with unconventional feedstuffs (Camelina sativa seeds; CS) rich in bioactive molecules such as polyunsaturated fatty acids, may prove a potential eco-efficient strategy to manipulate rumen microbiome towards efficiency. Forty-eight ewes were divided into four homogenous groups (n = 12) according to their fat-corrected milk yield (6%), body weight, and age, and were fed individually with concentrate, alfalfa hay, and wheat straw. The concentrate of the control group (CON) had no CS inclusion, whereas the treated groups were supplemented with CS at 60 (CS6), 110 (CS11), and 160 (CS16) g·kg−1 of concentrate, respectively. Rumen digesta was collected using an esophageal tube and then liquid and solid particles were separated using cheesecloth layers. An initial bacteriome screening using next-generation sequencing of 16S was followed by specific microbes targeting with a RT-qPCR platform, which unveiled the basic changes of the rumen microbiota under CS supplementation levels. The relative abundances of Archaea and methanogens were significantly reduced in the solid particles of CS11 and CS16. Furthermore, the relative abundance of Protozoa was significantly increased in both rumen fluid and solid particles of the CS6, whereas that of Fungi was significantly reduced in the rumen particle of the CS16. In rumen fluid, the relative abundance of Fibrobacter succinogens and Ruminobacter amylophilus were significantly increased in the CS6 and CS11, respectively. In the solid particles of the CS11, the relative abundance of Ruminococcus flavefaciens was significantly reduced, whereas those of Butyrivibrio proteoclasticus and Ruminobacter amylophilus were significantly increased. Additionally, the relative abundance of Selenomonas ruminantium was significantly increased in both CS11 and CS16. Consequently, the highest CS content in the concentrate reduced the relative abundance of methanogens without inducing radical changes in rumen microorganisms that could impair ruminal fermentation and ewes’ performance.

Dataset Information

Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing.

Publications

Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing.

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