ABSTRACT
Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence-specific endonucleases to generate DNA double strand breaks (DSBs) at user-specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non-homologous end joining (NHEJ)-mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology-directed repair (HDR)-mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2-1a (FAD2-1a) locus of embryogenic cells in tissue culture. We then describe ZFN- and NHEJ-mediated, targeted integration of two different multigene donors to the FAD2-1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T1 progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ-integrated donor were perfect or near-perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ-mediated targeted insertions of multigene donors at an endogenous genomic locus.
Subject(s)
DNA End-Joining Repair , Gene Editing , Gene Targeting , Glycine max/genetics , Zinc Finger Nucleases/metabolism , DNA Breaks, Double-Stranded , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Somatic Embryogenesis Techniques , Plants, Genetically Modified , Recombinational DNA Repair , Glycine max/embryology , Glycine max/enzymology , Transformation, Genetic , Transgenes , Zinc Finger Nucleases/geneticsABSTRACT
Sorghum (Sorghum bicolor (L.) Moench) is an important source for food, feed, and possesses many agronomic attributes attractive for a biofuels feedstock. A warm season crop originating from the semi-arid tropics, sorghum is relatively susceptible to both cold and freezing stress. Enhancing the ability of sorghum to tolerate cold and freezing offers a route to expand the acreage for production, and provides a potential drought avoidance strategy during flowering, an important parameter for protection of yield. Targeted perturbation of the signal transduction pathway, that is triggered by exposure to abiotic stress in plants, has been demonstrated in model systems as an avenue to augment tolerance. Calcium-dependent protein kinases (CDPKs) are key players in a plant's response to environmental assaults. To test the impact of modulating CDPK activity in sorghum as a means to enhanced abiotic stress tolerance, we introduced a constitutively expressed rice CDPK-7 (OsCDPK-7) gene construct. Sorghum transformants carrying this cassette, were not improved in cold or salt stress under the conditions tested. However, a lesion mimic phenotype and up-regulation of a number of pathogen related proteins, along with transcripts linked to photosynthesis were observed. These results demonstrate that modulating the Ca signaling cascade in planta via unregulated enhanced CDPK activity can lead to off-type effects likely due to the broadly integrated nature of these enzymes in signaling.
