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

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.

Keywords: biolistic transformation; gene targeting; genome editing; somatic embryogenesis; soybean; zinc finger nuclease.

Figure 1

Schematic of targeting constructs and target site. (a) HDR donor. hptII denotes the hygromycin phosphotransferase gene and its regulatory elements; HR1 and HR2 are 1 kb homology arms. (b) FAD2‐1a locus, showing the relative position of the FAD2‐1a coding sequence (white arrow), ZFN target site (grey diamond) and homology arms ( HR1 and HR2). (c) ZFN expression construct, showing the configuration of zinc finger arrays (ZF1 and ZF2), FokI domains and 2A ribosomal stutter sequence. (d) 7.1 kb NHEJ donor before and after linearization at the ZFN target site. RFP: red fluorescent protein gene. (e) 16.2 kb, four‐gene donor, before and after linearization at the ZFN target site. LP, landing pad; dgt‐28, glyphosate tolerance marker; aad‐1, 2,4‐D tolerance marker; dsm‐2, glufosinate tolerance marker. The thin, black lines and half‐diamonds flanking the transgenes in (d) and (e) represent vector backbone and ZFN monomer‐binding sites respectively.

Figure 2

HDR‐ and NHEJ‐mediated targeting in embryogenic tissue culture. (a) Representative stringent junction PCR results on 24 candidate events each from HDR‐based targeting experiments (top row of each gel) or NHEJ‐based targeting experiments (bottom row of each gel). 5′, 3′, forward and reverse are all with respect to the FAD2‐1a target locus. Black arrowheads indicate the expected size of an amplicon from a fully intact donor. HDR‐based targeting is not anticipated to give rise to donor integration in reverse orientation. (b) Circos plots (http://circos.ca) showing NGS read coverage across the donor (blue histogram above the corresponding section of the genetic map) and deduced donor integration sites (arcs) for a single copy (left) and a complex multicopy transgenic event (right). The red arc indicates the desired insertion site at FAD2‐1a; the black arcs indicate off‐target insertions.

Figure 3

NHEJ‐mediated targeting in somatic embryogenesis‐derived mature plants. (a) Position of overlapping, genome‐anchored junction amplicons in relation to a fully intact, 7.1 kb donor targeted to FAD2‐1a, with overlapping Sanger sequencing reads from a representative targeted event (event F2.B7). Grey diamonds represent the ZFN target site, duplicated in the case of a perfectly integrated donor. (b) Visualized DNA amplicons from the same representative sample shown in (a). Expected product sizes are marked with a black arrowhead. (c) Sanger sequencing‐derived chromatograms corresponding to the genome‐donor junctions of a representative targeted event (event Y9.C6). The sequence of the perfect junction is GAAAGG. This event has a single nucleotide substitution (A to G) at the 5′ genome‐donor junction and a perfect 3′ donor‐genome junction. (d) Sequences of both genome‐donor junctions from all nine fully intact targeted events obtained. The sequences of the individual ZFN monomer‐binding sites are shown in red.

Figure 4

NHEJ‐mediated targeting and Mendelian transmission of a 16.2 kb, four‐gene donor. (a) Position of genome‐anchored junction amplicons (1‐4) and internal amplicons (5‐8) in relation to a fully intact 16.2 kb donor targeted to FAD2‐1a in either forward orientation (top) or reverse orientation (bottom). (b) Detection of junction amplicons and internal amplicons in three independent, fully intact, targeted T₀ events. The three events are shown in the same order in each gel, but not individually labelled for clarity. Detection of amplicons 1 and 2 in event 2 indicates that the donor has integrated in the forward orientation, whereas detection of amplicons 3 and 4 in events 1 and 3 indicates that the donor has integrated in reverse orientation. All three events yielded all four internal donor amplicons. (c) Targeted insertions were faithfully transmitted to T₁ progeny. Summarized are the results of junction PCR and qPCR for all four donor‐associated genes for 22 T₁ progeny of the three T₀ events shown in (b). T₀ events 1 and 3 produced a total of five viable T₁ progeny each, the analytical results of which are shown. The co‐segregation of all four qPCR signals and both junction amplicons is nearly perfect.