Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed ( L.) using CRISPR-Cas9 system.

is one of the most important oilseed crops, and can supply considerable amounts of edible oil as well as provide raw materials for the production of biodiesel in the biotechnology industry. Lysophosphatidic acid acyltransferase (LPAT), a key enzyme in the Kennedy pathway, catalyses fatty acid chains into 3-phosphoglycerate and promotes further production of oil in the form of triacylglycerol. However, because is an allotetraploid with two subgenomes, the precise genes which involved in oil production remain unclear due to the intractability of efficiently knocking out all copies with high genetic redundancy. Therefore, a robust gene editing technology is necessary for gene function analysis.An efficient gene editing technology was developed for the allotetraploid plant using the CRISPR-Cas9 system. Previous studies showed poor results in either on-target or off-target activity in . In the present study, four single-gRNAs and two multi-gRNAs were deliberately designed from the conserved coding regions of which has seven homologous genes, and , which has four homologous genes. The mutation frequency was found to range from 17 to 68%, while no mutation was observed in the putative off-target sites. The seeds of the 2/5 mutant were wizened and showed enlarged oil bodies, disrupted distribution of protein bodies and increased accumulation of starch in mature seeds. The oil content decreased, with an average decrease of 32% for lines and 29% for lines in single-gRNA knockout lines, and a decline of 24% for mutant lines (i.e., g123) and 39% for / double mutant lines (i.e., g134) in multi-gRNA knockout lines.Seven homologous genes and four homologous genes were cleaved completely using the CRISPR-Cas9 system, which indicated that it is effective for editing all homologous genes in allotetraploid rapeseed, despite the relatively low sequence identities of both gene families. The size of the oil bodies increased significantly while the oil content decreased, confirming that and play a role in oil biosynthesis. The present study lays a foundation for further oil production improvement in oilseed crop species.