CRISPR/Cas9-mediated editing of double loci of BnFAD2 increased the seed oleic acid content of rapeseed (Brassica napus L.)

Abstract

Seed oleic acid is an important quality trait sought in rapeseed breeding programs. Many methods exist to increase seed oleic acid content, such as the CRISPR/Cas9-mediated genome editing system, yet there is no report on seed oleic acid content improvement via this system’s precise editing of the double loci of BnFAD2. Here, a precise CRISPR/Cas9-mediated genome editing of the encoded double loci (A5 and C5) of BnFAD2 was established. The results demonstrated high efficiency of regeneration and transformation, with the rapeseed genotype screened in ratios of 20.18% and 85.46%, respectively. The total editing efficiency was 64.35%, whereas the single locus- and double locus-edited ratios were 21.58% and 78.42%, respectively. The relative proportion of oleic acid with other fatty acids in seed oil of mutants was significantly higher for those that underwent the editing on A5 copy than that on C5 copy, but it was still less than 80%. For double locus-edited mutants, their relative proportion of oleic acid was more than 85% in the T1 and T4 generations. A comparison of the sequences between the double locus-edited mutants and reference showed that no transgenic border sequences were detected from the transformed vector. Analysis of the BnFAD2 sequence on A5 and C5 at the mutated locus of double loci mutants uncovered evidence for base deletion and insertion, and combination. Further, no editing issue of FAD2 on the copy of A1 was detected on the three targeted editing regions. Seed yield, yield component, oil content, and relative proportion of oleic acid between one selected double loci-edited mutant and wild type were also compared. These results showed that although the number of siliques per plant of the wild type was significantly higher than those of the mutant, the differences in seed yield and oil content were not significant between them, albeit with the mutant having a markedly higher relative proportion of oleic acid. Altogether, our results confirmed that the established CRISPR/Cas9-mediated genome editing of double loci (A5 and C5) of the BnFAD2 can precisely edit the targeted genes, thereby enhancing the seed oleic acid content to a far greater extent than can a single locus-editing system.

Environmental Risk Assessment Scenarios of Specific NGT Applications in <em>Brassicaceae</em> Oilseed Plants

“…The PUFA content of seeds is mainly reduced by knocking out FAD genes (Jiang et al, 2017;Morineau et al, 2017;Okuzaki et al, 2018;Huang et al, 2020;Jarvis et al, 2021;Lee et al, 2021;Liu et al, 2022;Shi et al, 2022). Another study successfully decreased the PUFA content by knocking out CIPK9 genes (Wang et al, 2023).…”

“…For example, oleic acid contents were elevated by knocking out FAD genes (Jiang et al, 2017;Morineau et al, 2017;Okuzaki et al, 2018;Huang et al, 2020;Jarvis et al, 2021;Lee et al, 2021;Liu et al, 2022;Shi et al, 2022).…”

CRISPR-mediated technology for seed oil improvement in rapeseed: Challenges and future perspectives

“…It was discovered that the impact of various mutation types at BnFAD2 alleles on oleic acid levels varied, indicating a potential for fatty acid level manipulation by precise mutation at specific region of a gene ( Huang et al., 2020 ). A recent study on BnFAD2 showed that the CRISPR/Cas9-mediated genome editing of the BnFAD2 ’s double loci (A5 and C5) can precisely edit the targeted genes, increasing the seed’s oleic acid content to a much greater extent than can a single locus-editing technique ( Liu H. et al., 2022 ). Recent findings also reveal that induced mutations in the BnFAD2 genes by CRISPR/Cas9-mediated editing has produce high oleic acid germplasm from the CY2 cultivar ( Shi et al., 2022 ).…”

Modification of Fatty Acid Profile and Oil Contents Using Gene Editing in Oilseed Crops for a Changing Climate

“… 64 Thlaspi arvense (pennycress) FAD2 , ROD ( reduced oleate desaturase ) Fad2 or fad2 fae1 could double oleic acid contents 65 Arachis hypogea (peanut) FAD2 Seed-specific down regulation of gene led to increased oleic acid content 66 Oryza sativa (rice) OsFAD2–1 Expression of Os FAD2–1 was disrupted which doubled oleic acid contents 67 Brassica napus (rapeseed) fad2_Aa Increase in high oleic acid contents 68 BnaFAD2 Oleic acid contents increased from 65% to 80%. Mutation in BnaFAD2 in A5 genome caused more significant fatty acid profile changes 69 BnFAD2 and BnFAE1 Increase oleic acid (70–80%) with decreased erucic acid 70 BnFAD2 Poor agronomic performance but high oleic acid content while oil content remain constant 71 Glycine max (soybean) GmFAD2 Oleic acid 80% with corresponding decrease in linoleic acid to 1.3% 72 Oleic acid content increased 11% to 40–50% in fad2-1a and fad2-1b mutants, and to 85% in the fad2-1a/fad2 73 Arachis hypogea (peanut) ahFAD2A High oleic acid content 74 Elaeis guineensis (oil palm) EgFAD2 and EgPAT High oleic acid and stearic acid palm oil …”