CRISPR/Cas9-mediated genome-editing for enhancing the total shoot lipid content in alfalfa

Lack of statutory lipid content in vegetative tissues of forages leads to higher methane emissions primarily due to the reactions of the methanogens in the fermentation process and higher intake by ruminants. While this can be overcome through the supplementation of lipids in the ruminants' diet, this is costly and impractical. Alfalfa (Medicago sativa L.) is a notable forage crop in terms of nutritional quality and export value; however, it contains only 2-4% lipid content in its aboveground vegetative biomass on a dry matter basis. As such, the aim of this study is to enhance total shoot lipid content (TSLC) in alfalfa by disrupting PEROXISOMAL TRANSPORTER 1 (PXA1) and SUGAR DEPENDENT 1 (SDP1) genes using CRISPR/Cas9-mediated genome editing. Three guide RNAs (gRNAs) were designed for both genes separately, which were then inserted into a background vector and introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Gene editing frequency droplet digital PCR (GEF-ddPCR) assays and Sanger sequencing were used to confirm the existence of mutations at the target sites. Eight unique PXA1-edited genotypes have been identified with GEF ranging from 25% to 75% through ddPCR. Similarly, 12 SDP-edited genotypes with GEF ranging from 25% to 75% have been confirmed using ddPCR and T7E1 endonuclease assay. No mosaics were identified in any of the above-identified genotypes. Preliminary shoot lipid analyses indicated that PXA1-edited genotypes possessed a small but significant 8.9% relative increase in TSLC compared to wild-type controls. Furthermore, fatty acid compositional analyses demonstrated that SDP1-edited genotypes exhibited a significant increase in linoleic acid (18:2) compared to controls. The application of this gene editing method to develop new germplasm with high TSLC could contribute to a reduction in greenhouse gas emissions from ruminant production systems in the future.