Zinc finger nuclease-mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non-homologous end joining.

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.

A short synthetic MAR positively affects transgene expression in rice and Arabidopsis.

Matrix Attachment Regions (MARs) are DNA elements that are thought to influence gene expression by anchoring active chromatin domains to the nuclear matrix. When flanking a construct in transgenic plants, MARs could be useful for enhancing transgene expression. Naturally occurring MARs have a number of sequence features and DNA elements in common, and using different subsets of these sequence elements, three independent synthetic MARs were created. Although short, these MARs were able to bind nuclear scaffold preparations with an affinity equal to or greater than naturally occurring plant MARs. One synthetic MAR was extensively tested for its effect on transgene expression, using different MAR orientations, plant promoters, transformation methods and plant species. This MAR was able to increase average transgene expression and produced integration patterns of lower complexity. These data show the potential of making well defined synthetic MARs and using them to improve transgene expression.