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

• DOI: 10.1111/pbi.13012
• Publication Date: 2018-09-16T13:25:17Z
• Authors: Nicholas D. Bonawitz; W. Michael Ainley; Asuka Itaya; Sivarama R. Chenn...; Tobias Cicak; Katherine Effinger; Ke Jiang; Tejinder Kumar Mall; Pradeep Reddy Marri; J. Pon Samuel; Nagesh Sardesai; Matthew Simpson; Otto Folkerts; Rodrigo Sarria; Steven R. Webb; Delkin O. Gonzalez; Daina H. Simmonds; Dayakar R. Pareddy
• Keywords: Gene Editing; Plant Somatic Embryogenesis Techniques; 0301 basic medicine; 2. Zero hunger; DNA End-Joining Repair; Glycine max; Recombinational DNA Repair; Plants, Genetically Modified; Zinc Finger Nucleases; 03 medical and health sciences; Transformation, Genetic; Gene Targeting; DNA Breaks, Double-Stranded; Transgenes; Research Articles; Plant Proteins

SummaryEmerging 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.