A trait stacking system via intra-genomic homologous recombination.

MAIN CONCLUSION: A gene targeting method has been developed, which allows the conversion of 'breeding stacks', containing unlinked transgenes into a 'molecular stack' and thereby circumventing the breeding challenges associated with transgene segregation. A gene targeting method has been developed for converting two unlinked trait loci into a single locus transgene stack. The method utilizes intra-genomic homologous recombination (IGHR) between stably integrated target and donor loci which share sequence homology and nuclease cleavage sites whereby the donor contains a promoterless herbicide resistance transgene. Upon crossing with a zinc finger nuclease (ZFN)-expressing plant, double-strand breaks (DSB) are created in both the stably integrated target and donor loci. DSBs flanking the donor locus result in intra-genomic mobilization of a promoterless selectable marker-containing donor sequence, which can be utilized as a template for homology-directed repair of a concomitant DSB at the target locus resulting in a functional selectable marker via nuclease-mediated cassette exchange (NMCE). The method was successfully demonstrated in maize using a glyphosate tolerance gene as a donor whereby up to 3.3 % of the resulting progeny embryos cultured on selection medium regenerated plants with the donor sequence integrated into the target locus. The process could be extended to multiple cycles of trait stacking by virtue of a unique intron sequence homology for NMCE between the target and the donor loci. This is the first report that describes NMCE via IGHR, thereby enabling trait stacking using conventional crossing.

Transgenic trait deployment using designed nucleases.

The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value-adding traits such as those associated with crop performance are expected to involve multiple transgenes. Random integration of transgenes not only results in unpredictable expression and potential unwanted side effects but stacking multiple, randomly integrated, independently segregating transgenes creates breeding challenges during introgression and product development. Designed nucleases enable the creation of targeted DNA double-strand breaks at specified genomic locations whereby repair can result in targeted transgene integration leading to precise alterations in DNA sequences for plant genome editing, including the targeting of a transgene to a genomic locus that supports high-level and stable transgene expression without interfering with resident gene function. In addition, targeted DNA integration via designed nucleases allows for the addition of transgenes into previously integrated transgenic loci to create stacked products. The currently reported frequencies of independently generated transgenic events obtained with site-specific transgene integration without the aid of selection for targeting are very low. A modular, positive selection-based gene targeting strategy has been developed involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected, over multiple cycles of sequential transformation. This, combined with the demonstration of intragenomic recombination following crossing of transgenic events that contain stably integrated donor and target DNA constructs with nuclease-expressing plants, points towards the future of trait stacking that is less dependent on high-efficiency transformation.

Genome editing in plants via designed zinc finger nucleases.

The ability to create DNA double-strand breaks (DSBs) at specified genomic locations, which then stimulate the cell's naturally occurring DNA repair processes, has introduced intriguing possibilities for genetic modification. Zinc finger nucleases (ZFNs) are designed restriction enzymes consisting of a nonspecific cleavage domain fused to sequence-specific DNA binding domains. ZFN-mediated DSB formation at endogenous genomic loci followed by error-prone non-homologous end joining (NHEJ) repair can result in gene-specific mutations via nucleotide base pair insertions or deletions. Similarly, specific DNA sequence modifications can be made by providing donor DNA templates homologous to sequences flanking the cleavage site via homology-directed repair (HDR). Targeted deletions of intervening DNA sequence can be obtained by ZFNs used to create concurrent DSBs. Site-specific transgene integration into ZFN-induced DSBs is possible via either NHEJ or HDR. Genome editing can be used to enhance our basic understanding of plant gene function as well as modify and improve crop plants. As with conventional plant transformation technology, the efficiency of genome editing is absolutely dependent on the ability to initiate, maintain, and regenerate plant cell and tissue cultures.

Trait stacking via targeted genome editing.

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.

Activation domains for controlling plant gene expression using designed transcription factors.

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.

Designed transcriptional regulators for trait development.

Development is largely controlled by proteins that regulate gene expression at the level of transcription. These regulatory proteins, the genes that control them, and the genes that they control, are organized in a hierarchical structure of complex interactions. Altering the expression of genes encoding regulatory proteins controlling critical nodes in this hierarchy has potential for dramatic phenotypic modification. Constitutive over-expression of genes encoding regulatory proteins in transgenic plants has resulted in agronomically interesting phenotypes along with developmental abnormalities. For trait development, the magnitude and timing of expression of genes encoding key regulatory proteins will need to be precisely controlled and targeted to specific cells and tissues at certain developmental timepoints. Such control is made possible by designed transcriptional regulators which are fusions of engineered DNA binding proteins and activator or repressor domains. Expression of genes encoding such designed transcriptional regulators enable the selective modulation of endogenous gene expression. Genes encoding proteins controlling regulatory networks are prime targets for up- or down-regulation via such designed transcriptional regulators.

Transcriptional activation of Brassica napus ß-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor.

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola ß-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.

Targeting DNA to a previously integrated transgenic locus using zinc finger nucleases.

Targeting exogenously supplied DNA to a predetermined location within a plant genome provides a powerful tool for basic studies of plant gene function and opens up some intriguing possibilities for crop improvement. The induction of double-strand DNA breaks at specific genomic loci via the use of designed zinc finger nucleases (ZFNs) allows for targeted transgene integration. Preintegrating a reporter construct containing a nonfunctional herbicide resistance gene flanked by ZFN binding sites results in a locus capable of being targeted. Retransformation with a corresponding ZFN-expressing cassette and a donor DNA with sequences homologous to the integrated construct and capable of functionalizing the herbicide resistance gene following site-specific integration results in targeted DNA addition. Targeted DNA integration can be confirmed in herbicide-resistant plant cells using PCR analysis.

Zinc finger nuclease-mediated transgene deletion.

A transgene, flanked by zinc finger nuclease (ZFN) cleavage sites, was deleted from a stably transformed plant by crossing it with a second plant expressing a corresponding ZFN gene. A target construct, containing a GUS reporter gene flanked by ZFN cleavage sites, a GFP reporter gene and a PAT selectable marker gene, was transformed into tobacco. Basta-resistant plants were regenerated and screened for GUS and GFP expression. A second construct, containing a ZFN gene driven by the constitutive CsVMV promoter and an HPT selectable marker gene, was also transformed into tobacco. Selected T(0) plants were grown to maturity and allowed to self-pollinate. Homozygous target plants, which expressed GUS and GFP, were crossed with homozygous ZFN plants, which expressed the ZFN gene. Numerous GUS-negative plants were observed among the hybrids with one particular cross displaying approximately 35% GUS-negative plants. Evidence for complete deletion of a 4.3 kb sequence comprising the GUS gene was obtained and sequence confirmed. Co-segregation in F(2) progenies of 'truncated' and 'intact' target sequences with expected reporter gene phenotypes were observed. Since ZFNs can be designed to bind and cleave a wide range of DNA sequences, these results constitute a general strategy for creating targeted gene deletions.

Whiskers-mediated maize transformation.

There has been rapid progress in recent years in extending gene transfer capabilities to include plant species that fall outside the normal host range of Agrobacterium. Methods that allow direct DNA delivery into plant cells have contributed significantly to this expanded capability. Whiskers treatment is one means of delivering macromolecules, including DNA, to plant cells. Using relatively simple equipment and inexpensive materials, whiskers-mediated transformation of maize is possible. A critical prerequisite, however, is the establishment and maintenance of embryogenic tissue cultures as a source of totipotent, transformation-competent cells. Within hours of agitation in the presence of silicon carbide whiskers and DNA, embryogenic maize tissue cultures display transient gene expression, providing evidence for DNA uptake. Using appropriate selectable marker genes, following in vitro selection on inhibitory levels of a corresponding selection agent, stably transgenic tissue cultures can be generated from which fertile plants can be recovered. The timeline from whiskers treatment of embryogenic maize tissue cultures to fertile seed recovery is approximately 9 months, which is competitive with other methods of maize transformation.

Targeted transgene integration in plant cells using designed zinc finger nucleases.

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.