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Plant & cell physiology

Simultaneous targeting of multiple gene homeologues to alter seed oil production in Camelina sativa.


PMID 28444368

Abstract

The ability to easily transform Camelina sativa with biosynthetic enzymes derived from other plants has made this oil seed crop an ideal platform for the production of unusual lipids valuable for different applications. However, in addition to expressing transgenic enzymes, the suppression of endogenous enzyme activity to reduce competition for common substrates or cofactors is also required to enhance the production of target compounds. As camelina possesses a relatively undifferentiated hexaploid genome, up to three gene homeologues can encode for any particular enzymatic activity, complicating efforts to alter endogenous biosynthetic pathways. New genome editing technologies such as that offered by the CRISPR/Cas system, offer the capability to introduce mutations into specifically targeted genomic sites. Here, by using a carefully designed guide RNA identical to all three homeologues, we demonstrate the ability of the CRISPR/Cas genome editing system to introduce mutations in all three CsDGAT1 or CsPDAT1 homeologous genes important for triacylglycerol (TAG) synthesis in developing seeds. Sequence analysis from transgenic T1 plants revealed that each CsDGAT1 or each CsPDAT1 homeologue was altered by multiple mutations, resulting in a genetic mosaic in the plants. Interestingly, seed harvested from both CsDGAT1- and CsPDAT1-targeted lines was often shrunken and wrinkled. Further, lipid analysis revealed that many lines produced seed with reduced oil content and altered fatty acid composition, consistent with the role of the targeted genes in seed oil biosynthesis. The CRISPR/Cas system therefore represents a useful method to efficiently alter endogenous biosynthetic pathways in polyploid species such as camelina.