Design of high-oleic tobacco (Nicotiana tabacum L.) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2–2

BACKGROUND: Tobacco seed oil could be used as an appropriate feedstock for biodiesel production. However, the high linoleic acid content of tobacco seed oil makes it susceptible to oxidation. Altering the fatty acid profile by increasing the content of oleic acid could improve the properties of biodiesel produced from tobacco seed oil. RESULTS: Four FAD2 genes, NtFAD2–1a, NtFAD2–1b, NtFAD2–2a, and NtFAD2–2b, were identified in allotetraploid tobacco genome. Phylogenetic analysis of protein sequences showed that NtFAD2–1a and NtFAD2–2a originated from N. tomentosiformis, while NtFAD2–1b and NtFAD2–2b from N. sylvestris. Expression analysis revealed that NtFAD2–2a and NtFAD2–2b transcripts were more abundant in developing seeds than in other tissues, while NtFAD2–1a and NtFAD2–1b showed low transcript levels in developing seed. Phylogenic analysis showed that NtFAD2–2a and NtFAD2–2b were seed-type FAD2 genes. Heterologous expression in yeast cells demonstrated that both NtFAD2–2a and NtFAD2–2b protein could introduce a double bond at the Δ(12) position of fatty acid chain. The fatty acid profile analysis of tobacco fad2–2 mutant seeds derived from CRISPR-Cas9 edited plants showed dramatic increase of oleic acid content from 11% to over 79%, whereas linoleic acid decreased from 72 to 7%. In addition, the fatty acid composition of leaf was not affected in fad2–2 mutant plants. CONCLUSION: Our data showed that knockout of seed-type FAD2 genes in tobacco could significantly increase the oleic acid content in seed oil. This research suggests that CRISPR-Cas9 system offers a rapid and highly efficient method in the tobacco seed lipid engineering programs.