Brief Communication Enhancing the accumulation of eicosapentaenoic acid and docosahexaenoic acid in transgenic Camelina through the CRISPR-Cas9 inactivation of the competing FAE1 pathwa

Han, L.

Haslam, R. P.

Lu, C.

Napier, J. A.

Silvestre, S.

English

PubMed

Han, Lihua

Haslam, Richard P.

Silvestre, Susana

Lu, Chaofu

Napier, Johnathan A.

PubMed Central

Enhancing the accumulation of eicosapentaenoic acid and docosahexaenoic acid in transgenic Camelina through the CRISPR‐Cas9 inactivation of the competing 
FAE1
 pathway

Rothamsted Repository

Patron:		Her	Majesty	The	Queen	 	 Rothamsted	Research	Harpenden,	Herts,	AL5	2JQ		Telephone:	+44	(0)1582	763133	Web:	http://www.rothamsted.ac.uk/			 			Rothamsted Research is a Company Limited by Guarantee Registered Office: as above.  Registered in England No. 2393175. Registered Charity No. 802038.  VAT No. 197 4201 51. Founded in 1843 by John Bennet Lawes.		Rothamsted Repository DownloadA - Papers appearing in refereed journalsHan, L., Haslam, R. P., Silvestre, S., Lu, C. and Napier, J. A. 2022. Enhancing the accumulation of eicosapentaenoic acid and docosahexaenoic acid in transgenic Camelina through the CRISPR-Cas9 inactivation of the competing FAE1 pathway. Plant Biotechnology Journal. https://doi.org/10.1111/pbi.13876 The publisher's version can be accessed at:• https://doi.org/10.1111/pbi.13876The output can be accessed at: https://repository.rothamsted.ac.uk/item/98974/enhancing-the-accumulation-of-eicosapentaenoic-acid-and-docosahexaenoic-acid-in-transgenic-camelina-through-the-crispr-cas9-inactivation-of-the-competing-fae1-pathway.© 20 June 2022, Please contact library@rothamsted.ac.uk for copyright queries.25/07/2022 15:32 repository.rothamsted.ac.uk library@rothamsted.ac.ukBrief CommunicationEnhancing the accumulation of eicosapentaenoic acid anddocosahexaenoic acid in transgenic Camelina through theCRISPR-Cas9 inactivation of the competing FAE1 pathwayLihua Han1, Richard P. Haslam1, Susana Silvestre1, Chaofu Lu2 and Johnathan A. Napier1,*1Plant Sciences Department, Rothamsted Research, Harpenden, Herts, UK2Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, Montana, USAReceived 11 April 2022;revised 8 June 2022;accepted 15 June 2022.*Correspondence (Tel +44 (0)1582 938136; emailjohnathan.napier@rothamsted.ac.uk)Keywords: metabolic engineering, Omega-3, Camelina, seed oil,gene editing, field trials.Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs),eicosapentaenoic acid (EPA; 20:5D5,8,11,14,17) and docosahex-aenoic acid (DHA; 22:6D4,7,10,13,16,19) are now accepted as beingessential components of a healthy, balanced diet (Napieret al., 2019; West et al., 2021). The wild capture fisheries thatsupply omega-3 fatty acids are at their maximum levels ofsustainable production; therefore, attempts to meet the growingdemands of an increasing population depend on alternativesources of fish oils (Tocher et al., 2019). Camelina sativa is anoilseed crop with high levels (>35%) of a-linolenic acid (ALA;18:3D9,12,15) and has reconstituted a biosynthetic pathway fromALA to synthesize both EPA and DHA in Camelina cv. Celineseeds by expressing heterologous desaturase and elongase genes,producing levels of EPA and DHA equivalent to those in marinefish oils, exemplified by the prototype line DHA2015.1 (abbrevi-ated to DHA1) accumulating over 25% n-3 LC-PUFAs (Figures S1and S2 (Petrie et al., 2014; Ruiz-Lopez et al., 2014). Field trials ofDHA1 in the UK, USA and Canada demonstrated the omega-3LC-PUFAs trait was stable in distinct geographical locations andagricultural environments (Han et al., 2020). In parallel, salmonfeeding trials and human dietary studies using DHA1 seed oilsboth demonstrated that these transgenic plant-derived oils couldserve as effective replacements for marine-derived fish oils(Betancor et al., 2018; West et al., 2021).Based on our observation that ALA is the endogenous C18precursor for seed omega-3 LC-PUFA production (Hanet al., 2020), we hypothesized that increasing the ALA pool canfurther enhance EPA/DHA accumulation in DHA1 Camelina. TheDHA1 construct already contains a D12 desaturase to drive theflux of fatty acids into PUFA biosynthesis (Figures S1 and S2).However, as a less obvious approach, we proposed using gene-edited fae1 mutants of Camelina. Camelina FAE1 functions incompetition with endogenous FAD2 D12 desaturase (whichdesaturates OA to produce linoleic acid (LA; 18:2D9,12)) for C18substrates, sequentially elongating oleic acid (OA; 18:1D9) togondoic acid 20:1D11 and then erucic acid 22:1D13. Previously,Ozseyhan et al. (2018) used CRISPR/cas9 to disrupt all threehomologues of FAE1 present in Camelina cv. Suneson (allo-hexaploid). Since the expression of FAE1 is restricted to develop-ing seeds, there is no impact of this mutation on other tissues.Within the seeds, ALA was increased from 36.9% in wildtype(WT) to 47.3% in fae1 mutant—this is in addition to thecomplete ablation of erucic acid, a fatty acid, which is consideredundesirable above a modest threshold (5% of total oil) in humanfoodstuffs. Pollen from the fae1 GE mutant (previously selfed tosegregate away from the CRISPR-Cas9 transgene and associatedDsRed marker) was crossed with the DHA1 line described in Hanet al. (2020), and the resulting F1 hybrid seeds were sown in thegreenhouse. Selected seeds with strong DsRed fluorescence(associated with DHA1; Figure S2) from individual F2 plants weredissected, and half the cotyledon tissue was used for fatty acidcomposition analysis by GC-FID, the other half germinated on 1/2MS nutrition media. Based on gas chromatographic analysis offatty acid methyl esters (FAMEs) data, only seeds showing a low20:1D11 content (<1.0%; Ozseyhan et al., 2018) were identifiedas the DHA1 and fae1 homozygous line (designated DHA1xfae1).Seeds from F3 plants were then sown alongside parental lines andWT as replicated plots in the field at the Rothamsted ExperimentalFarm (Harpenden, UK) on 23 May 2019, as permitted by Consent19/R8/01 from DEFRA. After harvesting on 18 September 2019,replicated seed FAMEs and TAG analysis were completed for theplots.GC analysis (Figure 1a) confirmed showed that in field-grownDHA1xfae1 seeds, the accumulation of 20:1D11 is as low as0.4%, consistent with the mutant background. Compared withparental line DHA1, the levels of palmitic acid (16:0) and stearicacid (18:0) were broadly the same, with the OA and LA levelsslightly decreased by 1% and 2.5%, respectively, whereas theALA level was increased from 17.0% in DHA1 to 21.5%DHA1xfae1. This confirmed that targeted mutagenesis by geneediting of the FAE1 genes in the DHA1 genetic background didnot affect the OA precursor metabolites but did alter thedownstream desaturation pathway, as predicted. Analysis ofthe total C20+ n-3 LC-PUFAs in DHA1xfae1 seeds showed littlechange in EPA compared with DHA1 and 9.1% compared with9.3%. However, the DHA level was modestly increased, fromPlease cite this article as: Han, L., Haslam, R.P., Silvestre, S., Lu, C. and Napier, J.A. (2022) Enhancing the accumulation of eicosapentaenoic acid anddocosahexaenoic acid in transgenic Camelina through the CRISPR-Cas9 inactivation of the competing FAE1 pathway. Plant Biotechnol J., https://doi.org/10.1111/pbi.13876.ª 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.This is an open access article under the terms of the Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the original work is properly cited.1Plant Biotechnology Journal (2022), pp. 1–3 doi: 10.1111/pbi.138769.7% in DHA1 to 12.6% in DHA1xfae1. Eicosatetraenoic acid(ETA; 20:4D8,11,14,17) and docosapentaenoic acid (DPA;22:5D7,10,13,16,19) levels were similarly enhanced, 1.3% and1.5%, respectively. In sum, total C20+ n-3 fatty acids contents(comprising ETA, EPA, DPA and DHA) were increased by 5.5%from 27.5% in DHA1 to 33.0% in DHA1xfae1. These datademonstrated that increasing the precursor ALA substrate can bean effective approach to boost n-3 LC-PUFAs production in theDHA1 line.Health studies in humans have reported that diets with anincreased omega-6 (n-6) to n-3 ratio are highly pro-thromboticand pro-inflammatory, and contribute to the prevalence ofatherosclerosis, obesity, diabetes and a wide range of inflamma-tion disorders (West et al., 2021). Therefore, we calculated thedifferent n-3 and n-6 parameters (Figure 1b, c; Figure S3). Thetotal C20+ n-6 fatty acids including dihomo-c-linolenic acid(DGLA; 20:3D8,11,14) and arachidonic acid (ARA; 20:4D5,8,11,14)remained similar, at 3.1% in DHA1 and 2.8% in DHA1xfae1,giving a ratio of C20+ (n-3/n-6) as 8.8 in DHA1 and 11.7 inDHA1xfae1. The total n-3 content was 47.5% in DHA1 and58.2% in DHA1xfae1, whereas the total n-6 content was 23.6%and 20.2%, respectively. Therefore, the ratio of total (n-3/n-6)was 2.0 in DHA1 and 2.9 in DHA1xfae1, which is also asignificant increase compared with 2.2 in the fae1 mutant and1.7 in both WT Celine and WT Suneson lines, indicating thatDHA1xfae1 seed fatty acids have a even better health benefitsthan those of DHA1, fae1 and WTs.Triacylglycerol (TAG) is the major storage lipid for fatty acids inthe seed, and it consists of a glycerol backbone onto which threefatty acids are esterified. The fatty acid composition of individualTAG species (described by number of carbons:number ofdesaturations) can vary, and WT camelina seed can have ~80different molecular species of TAG (Usher et al., 2017). We havepreviously shown how transgene-derived accumulation of EPA,DPA and DHA expands the repertoire of TAG species (Hanet al., 2020). A comparison (Figure S4) of the TAG species in theseeds of field-grown DHA1 and DHA1xfae1 compared to relevantparental lines (WT Celine, WT Suneson, fae1) illustrates a changein the TAG fingerprint of fae1, DHA1 and DHA1xfae1. The mostabundant TAGs in fae1 are C54 species with 3 to 9 unsaturations—combinations of ALA (18:3) and LA (18:2). There is an almostcomplete absence of any C56 TAG species in fae1. In the case ofDHA1 and DHA1xfae1, more novel TAG (C58 but inclusive ofC56 to C66) species are present. These novel TAG species areabsent from all the parental and WT backgrounds. Notable is theoccurrence of some DHA-specific TAG molecular species thatdiffer between DHA1 and DHA1xfae1 (e.g. 64:17 and 66:17) anda number of TAG species such as 58:8–58:12 that are moreabundant, indicative of elevated accumulations of long-chainpolyunsaturated fatty acids.In conclusion, the combination of CRISPR-Cas9 gene editing toinactivate the FAE1 pathway clearly results in a beneficial increasein the levels of EPA, DHA and other omega-3 LC-PUFAs intransgenic Camelina harbouring the DHA2015.1 transgenes. Inparticular, the fae1 mutant not only is devoid of C20+ monoun-saturated fatty acids (including the undesirable C22 erucic acid;Bach and Faure, 2010) but also has increased levels of omega-3fatty acids such as ALA. Our previous studies have indicated thatALA is the primary endogenous precursor fatty acid used to makeEPA and DHA, and our data here further confirm this. Thiscontrasts with Canola, where recent attempts to engineer theaccumulation of EPA and DHA result in the metabolism of theFigure 1 Omega-3 long-chain polyunsaturated fatty acids composition in Camelina sativa. Total seed fatty acids were analysed by GC-FID aftermethanolysis. (a) Mol % fatty composition for WT, fae1, DHA1 or DHA1xfae1 seeds. Note the absence of EPA and DHA in WT and fae1 seeds, and how thelevels generated by the DHA1 construct are elevated in the fae1 background. (b) The % composition of omega-3 (n-3) and omega-6 (n-6) fatty acids. (c)The ratio of n-3 to n-6 fatty acids.ª 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–3Lihua Han et al.2omega-6 precursor LA. In that respect, Canola is biased towardsthe synthesis of omega-6 fatty acids whereas Camelina is biasedtowards omega-3, therefore Canola requires an additionaltransgene-derived ‘push’ to direct the flux of fatty acids ontothe omega-3 track (discussed in Napier et al., 2019). It is alsonoteworthy that Canola already lacks the FAE1 activity, selectedby conventional plant breeding for the absence of erucic acid,which is present in parental varieties of Brassica napus seed oil.Collectively, these data suggest that Camelina is a superior hostfor the transgene-derived seed-specific synthesis of omega-3 LC-PUFAs such as EPA and DHA, and representing the first exampleof an environmental release of a GM + GE stack.AcknowledgementThe authors thank BBSRC (UK) for financial support underInstitute Strategic Programme Grant BBS/E/C/000I0420.Declaration of interestJAN acts as a scientific advisor for Yield10 Biosciences. The otherauthors have no interests to declare.Author contributionsExperimental data were planned and collected by LH, SS, RH andCL. Constructs were designed and built by LH and JN. All authorscontributed to the writing of this manuscript.ReferencesBach, L. and Faure, J.D. (2010) Role of very-long-chain fatty acids in plantdevelopment, when chain length does matter. C. R. Biol. 333, 361–370.Betancor, M.B., Li, K., Bucerzan, V.S., Sprague, M., Sayanova, O., Usher, S.,Han, L. et al. (2018) Oil from transgenic Camelina sativa containing over 25%n-3 long-chain PUFA as the major lipid source in feed for Atlantic salmon(Salmo salar). Br. J. Nutr. 119, 1378–1392.Han, L., Usher, S., Sandgrind, S., Hassall, K., Sayanova, O., Michaelson, L.V.,Haslam, R.P. et al. (2020) High level accumulation of EPA and DHA in field-grown transgenic camelina – a multi-territory evaluation of TAGaccumulation and heterogeneity. Plant Biotechnol. J. 18, 1–12.Napier, J.A., Olsen, R.E. and Tocher, D.R. (2019) Update on GM canola crops asnovel sources of omega-3 fish oils. Plant Biotechnol. J. 17, 703–705.Ozseyhan, M.E., Kang, J., Mu, X. and Lu, C. (2018) Mutagenesis of the FAE1genes significantly changes fatty acid composition in seeds of Camelinasativa. Plant Physiol. Biochem. 123, 1–7.Petrie, J.R., Shrestha, P., Belide, S., Kennedy, Y., Lester, G., Liu, Q. and Divi, U.K.(2014) Metabolic engineering Camelina sativa with fish oil-like levels of DHA.PLoS One, 9, e85061.Ruiz-Lopez, N., Haslam, R.P., Napier, J.A. and Sayanova, O. (2014) Successfulhigh-level accumulation of fish oil omega-3 long-chain polyunsaturated fattyacids in a transgenic oilseed crop. Plant J. 77, 198–208.Tocher, D.R., Betancor, M.B., Sprague, M., Olsen, R.E. and Napier, J.A. (2019)Omega-3 long-chain polyunsaturated fatty acids, EPA and DHA: bridging thegap between supply and demand. Nutrients, 11, 89.Usher, S., Han, L., Haslam, R.P., Michaelson, L.V., Sturtevant, D., Aziz, M. andChapman, K.D. (2017) Tailoring seed oil composition in the real world:optimising omega-3 long chain polyunsaturated fatty acid accumulation intransgenic Camelina sativa. Sci. Rep. 7, 6570.West, A.L., Miles, E.A., Lillycrop, K.A., Napier, J.A., Calder, P.C. and Burdge,G.C. (2021) Genetically modified plants are an alternative to oily fish forproviding n-3 polyunsaturated fatty acids in the human diet: a summary ofthe findings of a Biotechnology and Biological Sciences Research Councilfunded project. Nutr. Bull. 46, 60–68.Supporting informationAdditional supporting information may be found online in theSupporting Information section at the end of the article.Figure S1 Schematic representation of biosynthetic pathways.Figure S2 Schematic representation of the DHA2015.1construct.Figure S3 Unsaturation index of seed lipids.Figure S4 TAG profile for field-grown material.ª 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–3Gene editing to enhance the accumulation of EPA & DHA 3

Enhancing the accumulation of eicosapentaenoic acid and docosahexaenoic acid in transgenic Camelina through the CRISPR-Cas9 inactivation of the competing FAE1 pathway

Enhancing the accumulation of eicosapentaenoic acid and docosahexaenoic acid in transgenic Camelina through the CRISPR-Cas9 inactivation of the competing FAE1 pathway

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