Modifying Camelina Seed Oil Production Using CRISPR-Cas9 Gene Editing

Abstract

Camelina sativa, a member of the mustard family, is an increasingly studied oil seed crop with valuable agronomic traits that make it a potential source of biofuels in the future. Seed oil is composed mostly of triacylglycerol (TAG) which is synthesized by two major enzymes: diacylglycerol acyltransferase (DGAT1) and phospholipid: diacylglycerol acyltransferase (PDAT1). Acetyl-triacylglycerols (acetyl-TAG), an alternate triacylglycerol produced by Camelina, has desirable biofuel properties including reduced-viscosity and a low freezing point. Production of acetyl-TAG is competed by production of TAG by DGAT1 and PDAT1. The objective of this project is to induce mutations using clustered regularly interspaced palindromic repeats-associated protein 9 (CRISPR-Cas9) into the genes encoding for DGAT1 and PDAT1, inhibiting enzyme function to increase yield of acetyl-TAG within viable seeds that maintain these mutations in their germline. Camelina sativa has a hexaploid genome with three homeologs for each gene. Induced mutations could occur on some, none, or all three gene loci. This study uses gel electrophoresis to identify deletions within CsDGAT1 and CsPDAT1 gene loci and observe continuation of mutations between first- and second-generation plants. Ninety-nine second-generation (T2) Camelina plants were grown including wild- type and DGAT1-CRISPR mutant lines. Only one line with a deletion mutation was identified. However, this plant possessed a diseased phenotype, having stunted growth with sickly, wrinkled and spotted leaves. One possibility is that off-target effects of Cas9 that caused unwanted mutations leading to the defective phenotype. Future work should aim to screen for deletions in additional Camelina plants and in plants of diploid genomes like Pennycress.