Flax is an important crop used for oil and fiber production. Although genetic engineering has been possible in flax, it is not commonly used to produce cultivars. However, the use of genome editing technology, which can produce site-specific mutations without introducing foreign genes, may be a valuable tool for creating elite cultivars that can be easily cultivated. The purpose of this study was to investigate the potential of genome editing in flax by establishing the clustered regularly interspaced short palindromic repeats (CR ISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) genome editing system using the phytoene desaturase (PDS) gene, which produces albino mutants that are easily identifiable. Four sgRNAs were designed from two PDS genes of Flax (LuPDS1 and LuPDS2), and CRISPR-Cas9 genome editing vectors were constructed. After gene transformation, albino phenotypes were observed in transformed callus and regenerated plantlets on selection media. Polymerase chain reaction (PCR) amplification and sequencing of the PDS genes revealed deletions and insertions in the albino tissues, indicating successful editing of the PDS genes. Potential off-target sites were analyzed, but no off-target mutations were found, indicating the specificity of the CRISPR-Cas9 system. The establishment of a flax genome editing system using the CRISPR-Cas9 technology opens up new possibilities for the genetic engineering of flax. This study demonstrates the potential of genome editing in creating elite cultivars that can be easily cultivated, which can have significant implications for the flax industry.
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References
1.
SimopoulosAP. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed Pharmacother, 2002; 56(8):365–379; doi: 10.1016/s0753-3322(02)00253-6
2.
ThompsonLU, ChenJM, LiT, et al.Dietary flaxseed alters tumor biological markers in postmenopausal breast cancer. Clin Cancer Res, 2005; 11(10):3828–3835; doi: 10.1158/1078-0432.CCR-04-2326
Lorenc-KukułaK, ZukM, KulmaA, et al.Engineering flax with the GT family 1 Solanum sogarandinum Glycosyltransferase SsGT1 confers increased resistance to fusarium infection. J Agric Food Chem, 2009; 57(15):6698–6705; doi: 10.1021/jf900833k
8.
QiuX, HongHP, DatlaN, et al.Expression of borage delta6 desaturase in Saccharomyces cerevisiae and oilseed crops. Can J Bot, 2002; 80(1):42–49; doi: 10.1139/b01-130
9.
ZukM, PreschaA, StryczewskaM, et al.Engineering flax plants to increase their antioxidant capacity and improve oil composition and stability. J Agric Food Chem, 2012; 60(19):5003–5012; doi: 10.1021/jf300421m
10.
VrbováM, KotrbaP, HoráčekJ, et al.Enhanced accumulation of cadmium in Linum usitatissimum L. plants due to overproduction of metallothionein α-domain as a fusion to β-glucuronidase protein. Plant Cell Tiss Organ Cult, 2013; 112(3):321–330; doi: 10.1007/s11240-012-0239-1
11.
RazzaqA, SaleemF, KanwalM, et al.Modern trends in plant genome editing: An inclusive review of the CRISPR/Cas9 toolbox. Int J Mol Sci, 2019; 20(16):4045; doi: 10.3390/ijms20164045
12.
NishitaniC, HiraiN, KomoriS, et al.Efficient genome editing in apple using a CRISPR/Cas9 system. Sci Rep, 2016; 6:31481; doi: 10.1038/srep31481
13.
NakajimaI, BanY, AzumaA, et al.CRISPR/Cas9-mediated targeted mutagenesis in grape. PLoS One, 2017; 12(5):e0177966; doi: 10.1371/journal.pone.0177966
14.
OdipioJ, AlicaiT, IngelbrechtI, et al.Efficient CRISPR/Cas9 genome editing of phytoene desaturase in Cassava. Front Plant Sci, 2017; 8:1780; doi: 10.3389/fpls.2017.01780
15.
FanD, LiuT, LiC, et al.Efficient CRISPR/Cas9-mediated targeted Mutagenesis in Populus in the first generation. Sci Rep, 2015; 5:12217; doi: 10.1038/srep12217
16.
FengZ, ZhangB, DingW, et al.Efficient genome editing in plants using a CRISPR/Cas system. Cell Res., 2013; 23(10):1229–1232; doi: 10.1038/cr.2013.114
17.
JiaH, WangN. Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One, 2014; 9(4):e93806; doi: 10.1371/journal.pone.0093806
18.
MengY, HouY, WangH, et al.Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula. Plant Cell Rep, 2017; 36(2):371–374; doi: 10.1007/s00299-016-2069-9
19.
ZhangB, YangX, YangC, et al.Exploiting the CRISPR/Cas9 system for targeted genome mutagenesis in Petunia. Sci Rep, 2016; 6:20315; doi: 10.1038/srep20315
20.
XingHL, DongL, WangZP, et al.A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol, 2014; 14:327; doi: 10.1186/s12870-014-0327-y
21.
LudvíkováM, GrigaM. Transgenic flax/linseed (Linum usitatissimum L.) – expectations and reality. Czech J Genet Plant Breed, 2015; 51(4):123–141; doi: 10.17221/104/2015-cjgpb
22.
SauerNJ, Narvaez-VasquezJ, MozorukJ, et al.Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in Plants. Plant Physiol., 2016; 170(4):1917–1928; doi: 10.1104/pp.15.01696
23.
MoerschellRP, TsunasawaS, ShermanF. Transformation of yeast with synthetic oligonucleotides. Proc Natl Acad Sci U S A, 1988; 85(2):524–528; doi: 10.1073/pnas.85.2.524
24.
YoonK, Cole-StraussA, KmiecEB. Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide. Proc Natl Acad Sci U S A, 1996; 93(5):2071–2076; doi: 10.1073/pnas.93.5.2071
25.
BeethamPR, KippPB, SawyckyXL, et al.A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. Proc Natl Acad Sci U S A, 1999; 96(15):8774–8778; doi: 10.1073/pnas.96.15.8774
26.
ZhuT, PetersonDJ, TaglianiL, et al.Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proc Natl Acad Sci U S A, 1999; 96(15):8768–8773; doi: 10.1073/pnas.96.15.8768
27.
AartsM, DekkerM, de VriesS, et al.Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells. Nucleic Acids Res, 2006; 34(21):e147; doi: 10.1093/nar/gkl896
28.
SauerNJ, MozorukJ, MillerRB, et al.Oligonucleotide-directed mutagenesis for precision gene editing. Plant Biotechnol J, 2016; 14(2):496–502; doi: 10.1111/pbi.12496
29.
SahaD, MukherjeeP, DuttaS, et al.Genomic insights into HSFs as candidate genes for high-temperature stress adaptation and gene editing with minimal off-target effects in flax. Sci Rep, 2019; 9(1):5581; doi: 10.1038/s41598-019-41936-1
30.
CharrierA, VergneE, DoussetN, et al.Efficient targeted mutagenesis in apple and first time edition of pear using the CRISPR/Cas9 System. Front Plant Sci, 2019; 10:40; doi: 10.3389/fpls.2019.00040
31.
WangX, TuM, WangD, et al.CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation. Plant Biotechnol J, 2018; 16(4):844–855; doi: 10.1111/pbi.12832
32.
PengA, ChenS, LeiT, et al.Engineering canker-resistant plants through CRISPR/Cas9 targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnol J., 2017; 15(12):1509–1519; doi: 10.1111/pbi.12733
33.
TianS, JiangL, GaoQ, et al.Efficient CRISPR/Cas9 based gene knockout in watermelon. Plant Cell Rep., 2017; 36(3):399–406; doi: 10.1007/s00299-016-2089-5
34.
MaX, ZhuQ, ChenY, et al.CRISPR/Cas9 platforms for genome editing in plants: Developments and applications. Mol Plant, 2016; 9(7):961–974; doi: 10.1016/j.molp.2016.04.009
35.
HepburnAG, ClarkeLE, BlundyKS, et al.Nopaline Ti-plasmid, pTiT37, T-DNA insertions into a flax genome. J Mol Appl Genet, 1983; 2(2):211–224.
36.
RakouskýS, TejklováE, WiesnerI, et al.Hygromycin B - An alternative in flax transformant selection. Biologia Plant, 1999; 42(3):361–369; doi: 10.1023/A:1002457000944
37.
BortesiL, FischerR. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv, 2015; 33(1):41–52; doi: 10.1016/j.biotechadv.2014.12.006
38.
Lorenc-KukułaK, Wróbel-KwiatkowskaM, StarzyckiM, et al.Engineering flax with increased flavonoid content and thus Fusarium resistance. Physiol Mol Plant Pathol, 2007; 70(1–3):38–48; doi: 10.1016/j.pmpp.2007.05.005
39.
WangS, ZhangS, WangW, et al.Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Rep, 2015; 34(9):1473–1476; doi: 10.1007/s00299-015-1816-7
40.
AnderssonM, TuressonH, NicoliaA, et al.Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR/Cas9 expression in protoplasts. Plant Cell Rep, 2017; 36(1):117–128; doi: 10.1007/s00299-016-2062-3
41.
WangJ, WuH, ChenY, et al.Efficient CRISPR/Cas9 mediated gene editing in an interspecific hybrid poplar with a highly heterozygous genome. Front Plant Sci, 2020; 11:996; doi: 10.3389/fpls.2020.00996
42.
CaiY, ChenL, LiuX, et al.CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean. Plant Biotechnol J, 2018; 16(1):176–185; doi: 10.1111/pbi.12758
43.
MaX, ZhangQ, ZhuQ, et al.A Robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 2015; 8(8):1274–1284; doi: 10.1016/j.molp.2015.04.007
44.
TsaiSQ, ZhengZ, NguyenNT, et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol., 2015; 33(2):187–197; doi: 10.1038/nbt.3117
45.
GaoJ, WangG, MaS, et al.CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol, 2015; 87(1–2):99–110; doi: 10.1007/s11103-014-0263-0
46.
GalinouskyD, MokshinaN, PadvitskiT, et al.The toolbox for fiber flax breeding: A pipeline from gene expression to fiber quality. Front Genet, 2020; 11:589881; doi: 10.3389/fgene.2020.589881
47.
MorelloL, PydiuraN, GalinouskyD, et al.Flax tubulin and CesA superfamilies represent attractive and challenging targets for a variety of genome- and base-editing applications. Funct Integr Genomics, 2020; 20(1):163–176; doi: 10.1007/s10142-019-00667-2
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