Enhancement of Tobacco ( Nicotiana tabacum L.) Seed Lipid Content for Biodiesel Production by CRISPR-Cas9-Mediated Knockout of NtAn1

Tobacco ( L.) seed lipid is a promising non-edible feedstock for biodiesel production. In order to meet the increasing demand, achieving high seed lipid content is one of the major goals in tobacco seed production. The gene and its homologs negatively regulate seed lipid accumulation in and species. We speculated that manipulating the homolog genes of in tobacco could enhance the accumulation of seed lipid. In this present study, we found that the homolog genes in tobacco, and , were highly expressed in developing seed. Targeted mutagenesis of genes was created by the CRISPR-Cas9-based gene editing technology. Due to the defect of proanthocyanidin (PA) biosynthesis, mutant seeds showed the phenotype of a yellow seed coat. Seed lipid accumulation was enhanced by about 18 and 15% in two targeted mutant lines. Protein content was also significantly increased in mutant seeds. In addition, the seed yield-related traits were not affected by the targeted mutagenesis of genes. Thus, the overall lipid productivity of the knockout mutants was dramatically enhanced. The results in this present paper indicated that tobacco genes regulate both PAs and lipid accumulation in the process of seed development and that targeted mutagenesis of genes could generate a yellow-seeded tobacco variety with high lipid and protein content. Furthermore, the present results revealed that the CRISPR-Cas9 system could be employed in tobacco seed domestication for biodiesel feedstock production.