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. 2015 Aug 30;14(2):503–509. doi: 10.1111/pbi.12457

Transgenic trait deployment using designed nucleases

Joseph F Petolino 1,✉, Sandeep Kumar 1
PMCID: PMC11388940  PMID: 26332789

Summary

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.

Keywords: designed nuclease, trait stacking, position effect

Introduction

As the human population hurtles towards nine billion by 2050 and climate change continues to manifest itself in yet unforeseen ways, the need to improve agricultural productivity across broad environmental conditions has never been greater. It has been estimated that high‐quality food production will need to increase by 70% in this timeframe with reduced greenhouse gas emissions and improved water‐use efficiency (FAO, 2009). As such, no technological ‘stone’ should be left ‘unturned’.

The twentieth century saw tremendous advances in the ability to increase agricultural productivity via the use of new technology for the generation and deployment of genetically enhanced crops. For example, the use of natural and induced mutations combined with phenotypic selection led to the identification and subsequent utilization of numerous value‐added traits and the onset of the so‐called Green Revolution (Sigurbjornsson, 1971). The exploitation of heterosis via the use of hybrids increased yields dramatically in certain key crops, in particular maize (Crow, 1998). The use of tissue culture methods such as micropropagation which facilitated the massive multiplication of selected genotypes of numerous plant species (Debergh and Read, 1991); embryo rescue which allowed for the generation of previously incompatible crosses with wild relatives (Sharma et al., 1996); and microspore culture which improved breeding efficiency by rapid inbred line production (Ferrie and Caswell, 2011)—have all had a significant impact on plant improvement. In addition, molecular mapping of crop genomes and marker‐assisted selection allowed for rapid progress in breeding both qualitative and quantitative traits (Collard and Mackill, 2008). High‐throughput DNA analysis has resulted in the determination of complete genome sequences of several plant species (Edwards and Batley, 2010), and genomewide transcript, protein and metabolite data sets are being assembled and used to reveal functional relationships between genes and subsequent traits (Ficklin et al., 2010). Taken together, these advances have established a solid technological foundation for continued genetic enhancement of crops for global food production.

In the later part of the twentieth century, the application of recombinant DNA technology to plant biology ushered in the era of transgenic crops, thereby lifting the conventional crossing barriers between species and effectively expanding the pool of genes available for crop trait development (Raymond Park et al., 2011). The first commercially available transgenic crop was marketed in the mid‐1990s, and already today over 175 million hectares are planted globally (Clive, 2014). Pest resistance and herbicide tolerance have been the first traits to reach the marketplace; however, product offerings involving more complex traits, such as stress tolerance and nutrient‐use efficiency, and combinations of multiple traits are becoming the norm (Marra et al., 2010). Indeed, the need for products requiring ever more complex combinations of transgenes has resulted in new challenges for current and future trait deployment.

As new methods for genome interrogation and manipulation are developed and basic understanding of plant biology deepens, even more robust capabilities for genetic modification become available. Technology designed to introduce precise alterations in DNA sequences for plant genome editing is being brought to bear on the development and commercialization of transgenic crops. A key component of such technology is the designed nuclease which enables the creation of targeted DNA double‐strand breaks at specified genomic locations whereby repair can result in various types of genomic modifications, including targeted transgene integration (Shukla et al., 2009). Indeed, genome editing in plants using designed nucleases is already on the way towards deployment for crop improvement (Lusser et al., 2012). The current review focuses on how designed nucleases provide solutions to some of the challenges associated with the deployment of transgenic traits.

Challenges associated with transgenic trait deployment

The reliance on random transgene integration

Although copy number and sequence fidelity can have a dramatic influence on transgene expression stability, the chromosomal location into which DNA integrates is also a major source of uncertainty following transformation (Wilson et al., 1990). Neighbouring regulatory elements and the general epigenetic state of a particular chromosomal region can interact with the regulatory elements in the integrated transgene to influence expression in unpredictable ways (van Leeuwen et al., 2001). Moreover, some genomic loci are less tolerant to the integration of foreign DNA (Kumpatla et al., 1998). Attempts to buffer such transgene × locus interactions through the use of AT‐rich sequences that attach to the nuclear matrix have had mixed results (Mlynarova et al., 1996). Perhaps more significantly, nonspecific position effects, resulting from higher level chromosome architecture, can influence the behaviour of an incoming transgenic sequence (Matzke and Matzke, 1998).

Unpredictable transgene expression following random genome integration is not uncommon (Dean et al., 1988). For example, the loci of two stably expressed transgenes in tobacco were found to be flanked by host DNA containing AT‐rich regions in the vicinity of telomeres whereas unstably expressed transgenes were integrated into intercalary and paracentric locations (Iglesias et al., 1997). Similarly, analysis of the junctions between an integrated transgene and genomic DNA in rice stably expressing a reporter gene revealed the presence of AT‐rich repetitive sequences (Takano et al., 1997). The stochastic variability of transgene expression, attributed to the characteristics of the site of foreign DNA integration, is referred to as ‘chromosomal position effect’ (Alberts and Sternglanz, 1990). This unpredictable transgene behaviour resulting from ‘chromosomal position effect’ represents a significant challenge for the development and deployment of transgenic traits.

Another issue associated with the random integration of transgene sequences is the potential disruption of endogenous gene expression in genic regions via insertional mutagenesis, thereby creating undesired side effects. Transformation with promoter‐less reporter genes has resulted in the recovery of transgenic plants with reporter gene expression suggesting integration into transcriptionally active gene sequences (Lindsey et al., 1993). Similarly, transcriptional interference by highly expressed promoters on transgene constructs can potentially influence the expression of endogenous genes resulting in unanticipated side effects (Conner and Jacobs, 1999). These side effects compound the problem of lack of predictability in the performance of transgenically derived materials and further compromise the probability of successful product development.

Current transgenic trait development, which relies on the random integration of DNA sequences, requires the production of multiple, independently generated transgenic events with distinct integration sites and/or multiple random integrations that are then analysed for several generations in hopes of obtaining breeding materials that meet all of the demanding product requirements, including an appropriate level of expression, stability over generations and overall performance. Each event is defined by a DNA sequence integrated at a specific chromosomal location. As the overwhelming majority of transgenic events do not meet the rigorous performance requirements for trait development and commercialization, it is necessary to generate a large number to maximize the probability of success (Mumm and Walters, 2001). This need for large numbers of events and exhaustive performance assessment makes the current process of transgenic trait development time‐consuming, labour intensive and, as such, costly.

The need for transgene stacking

The majority of first‐generation transgenic products involved herbicide tolerance and insect resistance traits that required the introduction of relatively simple transgenic sequences such as single genes encoding herbicide insensitive target enzymes (Padgette et al., 1995) and/or insect toxins (Koziel et al., 1993). However, the current need for durable, broad‐spectrum control of weed and insect pests requires the integration of multiple transgenic sequences encoding gene products that provide multiple modes of action (Que et al., 2010). Moreover, recent advances in genomics and bioinformatics suggest that many important traits associated with crop performance and yield not only depend on a plurality of genes but also on their complex interactions with each other and with external stimuli (Dockter and Hansson, 2015). Developing and deploying products of this sort will demand the integration, expression and introgression of multiple transgenic sequences.

Delivering large, multigene constructs into a single genomic locus is not practical using current transformation methods (Dafny‐Yelin and Tzfira, 2007). As such, iterative strategies utilizing sequentially introduced transgenic sequences have typically been used (Halpin, 2005). Unfortunately, the paucity of available selectable markers for commercial use limits the number of cycles of retransformation that can be performed. This is particularly acute in long life cycle or asexually propagated species where conventional segregation is not an option. In addition, sequentially introduced, randomly integrated transgenes need to be combined using conventional crossing which can be time‐consuming and labour intensive.

As currently practiced, transgenic trait deployment requires the stacking of multiple, randomly integrated, independently segregating sequences. Typically, such stacking requires the crossing of multiple transgenic events containing sequences integrated at various genomic loci. This genetic complexity creates breeding challenges as multiple, unlinked transgenes must be sorted during the phases of introgression and product development (Figure 1a). If the minimum population size for an event to occur is loge (1 − P)/loge (1 − f) where P is the probability and f is the frequency of occurrence, then for six unlinked transgenic loci, since the frequency of occurrence of an F 2 individual homozygous at all six loci is 0.256 (2.4 × 10−4), one needs to screen over 19 000 F 2 individuals to virtually assure (P > 0.99) finding at least then one homozygous for all six transgenes. This requires a substantial investment in time and resources. Moreover, the linkage drag associated with the introgression of multiple genomic loci could negatively affect the overall performance of the finished product (Peleman and van der Voort, 2003).

Figure 1.

Figure 1

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Random vs. targeted transgene integration. (a) Multiple transgenes randomly integrated into different genomic locations segregate independently. (b) Multiple transgenes targeted to the same genomic location co‐segregate. Vertical bars represent chromosomes, and horizontal black lines represent individual transgenes.

Deploying transgenic traits with designed nucleases

Site‐specific transgene integration

The creation of sequence‐specific double‐strand breaks using designed nucleases which stimulate the cell's DNA repair machinery has led to the ability to integrate transgenes into specified genomic loci (Moehle et al., 2007). Several platforms for generating designed nucleases capable of creating targeted double‐strand breaks have been developed including zinc finger nucleases (Kim et al., 1996), meganucleases (Epinat et al., 2003), transcription activator‐like effector nucleases (Christian et al., 2010) and clustered, regularly interspaced, short palindromic repeat RNA‐guided nucleases (Wiedenheft et al., 2012). Using any of these designed nucleases, exogenously supplied DNA sequences can be integrated into the genome as a by‐product of DNA repair at the targeted cleavage site. Depending on the target cell and the architecture of the donor DNA sequence, repair can be homology‐driven, involving stretches of related sequence that serve as a template for synthesis‐dependent strand annealing, or can involve nonhomologous end joining to unrelated sequences or microhomologies (Puchta et al., 1996). Either way, transgenes become stably integrated into prespecified genomic locations.

The ability to target DNA to predetermined genomic loci has been demonstrated using designed nucleases and donor DNA homologous to either pre‐integrated or native sequences. Targeting transgenes to pre‐integrated nuclease cleavage sites typically involves the correction of a selectable marker gene to allow for isolation and propagation of the rare targeted event (Cai et al., 2009; D'Halluin et al., 2008; Wright et al., 2005). Targeting a native genomic locus in maize was demonstrated using a promoter‐less, herbicide resistance gene as a donor DNA capable of ‘trapping’ an endogenous promoter sequence (Shukla et al., 2009). These results clearly show the way for enhanced precision with respect to the generation of transgenic plants.

Targeting transgene sequences to specific locations within the plant genome foreshadows the need to identify appropriate sites to support trait development. An ideal genomic integration site should allow high‐level and stable transgene expression without interfering with resident gene function. Such genomic regions, referred to as ‘safe harbours’, have been proposed to consist of certain characteristics that predict appropriate transgene behaviour and a reduced probability of unwanted side effects. For example, in human induced pluripotent stem cells, a ‘safe harbour’ is defined as being at least 50 kb from the 5′ end of any gene, 300 kb from any cancer‐related gene or micro‐RNA and outside a transcription unit or ultra‐conserved region (Bejerano et al., 2004). Integrating a beta‐globin transgene into these regions resulted in high‐level expression without perturbation of neighbouring gene function (Papapetrou et al., 2011). Similarly, sustainable reporter gene expression was observed without detectable disturbance of flanking gene expression following targeted integration into three different human HEK293 cell loci (Eyquem et al., 2013). The integration of transgene sequences into ‘safe harbours’ for crop trait development would be an attractive approach to obtaining consistent expression and avoiding potential negative effects on endogenous gene function. Availability of safe harbour loci for use in plant genome engineering would be highly desirable.

Cre recombinase‐mediated integration of reporter genes into pre‐integrated lox sites provided evidence for the importance of position effect to transgene expression (Chawla et al., 2006; Day et al., 2000). Single copy events of rice which contained no illegitimate transgene integration displayed consistent levels of reporter gene expression over three to four successive generations while multicopy events showed expression variation and transgene silencing (Chawla et al., 2006). Among transgenic events of tobacco harbouring a single copy of a reporter gene construct, a 10‐fold level of expression variability was observed among the various target loci (Day et al., 2000). At a given locus, roughly half of the events displayed the same level and pattern of reporter gene expression while the other half showed some signs of transgene silencing resulting from DNA methylation. Using a designed zinc finger nuclease, four independent insertions into the same engineered genomic location of maize resulted in similar expression of a selectable marker gene and significantly different expression when integrated into a different locus (Kumar et al., 2015). So although targeted transgene integration might not solve all of the issues associated with event‐to‐event variability, results suggest that targeting to a specific locus provides more stable and predictable expression when compared with random integration.

Trait stacking via sequential targeted transgene integration

In addition to the need for an appropriate level of transgene expression, stably inherited over generations and meeting rigorous performance standards, modern agriculture requires flexible transgene deployment to meet increasingly complex product needs for different geographies, including the mandatory management of pest resistance (Gould, 1998). Stacking newly developed transgenes with existing traits by crossing independently generated events is a commercial challenge. For example, SmartStax™ 1 maize is an eight‐trait stack which provides multiple modes of insect resistance for more effective and durable pest management (Storer et al., 2012). This product was co‐developed by Dow AgroSciences and Monsanto by combining transgenes from four independently generated events (Siebert et al., 2012). Its success suggests that the ability to combine, add, subtract and/or replace new and existing transgenes will become even more important for future product offerings.

Targeted DNA integration via designed nucleases allows for the addition of transgenes into a previously integrated transgenic locus to create a stacked product (Figure 1b). Using a designed meganuclease, two herbicide tolerance genes were integrated into a pre‐existing insect control locus following targeted cleavage and homology‐directed repair in cotton (D'Halluin et al., 2013). Targeted events were recovered at a frequency of 1.8% of all transformed callus, and all genes were inherited as a single genetic unit in subsequent generations and accumulated the expected protein products. Similarly, a pre‐integrated ‘trait landing pad’ comprising a zinc finger binding site flanked by sequences homologous to an incoming donor DNA allowed for the sequential integration of two herbicide resistance genes into the same genomic locus in maize (Ainley et al., 2013). Up to 5% of the events were targeted to the locus, and both herbicide resistance genes were shown to functionally co‐segregate. Both of these reports exemplify the power of site‐specific transgene integration for creating sequential transgene stacks.

Using current plant transformation methods, targeting frequencies on an event basis are typically in the range of 1%–5% (Petolino et al., 2015). This frequency can be dramatically improved using positive and/or negative selection‐based strategies whereby only targeted events are recovered (Shimatani et al., 2014). Recently, a modular, positive selection‐based strategy has been developed for use in maize trait stacking involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected potentially over multiple cycles of sequential transformation (Kumar et al., 2015). This approach involves a target locus containing a first set of genes created by either targeted integration into a ‘safe harbour’ locus or random integration (Figure 2a). The target locus also includes a ‘trait landing pad’ between the first set of genes and a first selectable marker gene. The promoter driving the selectable marker gene in the target locus contains a unique 500–1000 bp 3′ intron sequence flanked by the same nuclease recognition sequence as found in the ‘trait landing pad’. Following nuclease‐mediated cleavage in the target locus, the 3′ intron and ‘trait landing pad’ provide homology with an incoming donor DNA containing a promoter‐less, second selectable marker gene and a second ‘trait landing pad’ for future targeting. In this way, a second set of genes can be introduced into the target locus using selection and the first selectable marker is deleted, thereby becoming available for future use. In principle, this process could be repeated over multiple cycles of targeted transformation with the same selectable marker genes and nuclease recognition sequences being recycled with each round of gene stacking (Figure 2b).

Figure 2.

Figure 2

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Multigeneration gene targeting and stacking strategy. (a) Plant with a pre‐integrated ‘Target Locus 1’ containing ‘Gene Stack 1’ and ‘Trait Landing Pad 1’ (TLP 1) comprising a ‘Nuclease Cleavage Site (NCS 1, black diamond), generated using ‘Selectable Marker A’, is co‐transformed with ‘Donor 1’ containing ‘Selectable Marker B’ and ‘Gene Stack 2’ along with a construct containing a nuclease expression cassette capable of cleaving NCS 1. Following cleavage in ‘Target 1’, homology between the intron and TLP 1 leads to a functional ‘Selectable Marker B’. ‘Donor 1’ also contains ‘Trait Landing Pad 2’ (TLP 2) containing a second nuclease cleavage site, NCS 2, for subsequent targeting (grey diamond). (b) ‘Target 2’ can be co‐transformed by reusing ‘Selectable Marker A’ with ‘Donor 2’ containing ‘Gene Stack 3’ and ‘Trait Landing Pad‐3’ (TLP 3) for further targeting. In this way, multiple generations of sequential transgene stacking become possible.

Transgene deletion

In addition to adding or replacing transgenes to create new products, the removal of transgenic sequences is also an anticipated capability need for trait deployment. This is particularly germane with respect to selectable and/or reporter genes which, although necessary for selective cell proliferation and plant regeneration during stable transformation, are often unnecessary for commercial trait development (Hohn et al., 2001). Moreover, the excision of selectable marker genes from transgenic events allow for their reuse during sequential transformation (Hare and Chua, 2002). In addition, the excision of transgenic sequences in pollen to eliminate unwanted transgene migration has been proposed as a potential biocontainment strategy (Moon et al., 2010). Most work in this area has involved the use of recombinase‐based systems whereby recombination between pre‐integrated recognition sites results in deletion of intervening DNA sequences (Odell et al., 1990). In this way, transgenic sequences targeted for subsequent deletion, for example selectable marker genes, can be flanked by directly repeated recognition sites and excised following recombinase gene expression (Dale and Ow, 1991; Zhang et al., 2003).

Designed nucleases have also been successfully used to excise stably integrated transgenic sequences (Antunes et al., 2012; Petolino et al., 2010). A reporter gene flanked by nuclease cleavage sites was deleted from a stably transformed plant following crossing to a plant that expressed a corresponding nuclease. In tobacco, two concurrent double‐strand breaks mediated by a designed nuclease promoted the removal of 4.3 kb of intervening DNA sequence, including a complete reporter gene cassette (Petolino et al., 2010). Similarly, in Arabidopsis a synthetic homing endonuclease gene under the control of a heat‐inducible promoter excised a selectable marker gene flanked by recognition sites following heat treatment (Antunes et al., 2012). The ability to delete even larger chromosomal sequences has been demonstrated in rice using nucleases designed to cleave endogenous genomic loci. Large deletions (>100 kb) containing a cluster of five phytoalexin biosynthetic genes were observed following concurrent cleavage at two genomic loci (Zhou et al., 2014). Similarly, ∼9 Mb deletions were obtained in Arabidopsis following nuclease cleavage in a cluster of tandemly arrayed gene sequences (Qi et al., 2013). Unlike recombinases which require the pre‐integration of recognition sites flanking the sequence targeted for deletion, being able to design nucleases to cleave virtually any DNA sequence offers a greater degree of flexibility with respect to transgene removal options.

Intragenomic recombination

Although the induction of sequence‐specific double‐strand DNA breaks has allowed for genome editing by co‐delivering donor DNA and nuclease‐encoding sequences, the frequency of targeted transgene integration is rather low compared to random integration (Petolino et al., 2015). The need to simultaneously deliver donor DNA and nuclease‐encoding sequences to large numbers of plant cells is particularly problematic in all but a few plant species that have a robust transformation capability. A solution to this problem involved creating transgenic events with all of the required editing reagents stably integrated into the genome. For example, in Drosophila, a donor sequence containing site‐specific recombinase and nuclease sites produced an extrachromosomal DNA capable of recombining with a target sequence via homology‐directed repair following recombinase and nuclease gene expression (Rong and Golic, 2000). In other words, a randomly integrated transgene flanked by recombinase sites could be liberated as a circular DNA, linearized via nuclease cleavage and targeted to a specific locus via intragenomic recombination through a homology‐directed repair mechanism. In Arabidopsis, expression of a site‐specific nuclease that cleaved both a target locus and an integrated donor resulting in an excised DNA capable of facilitating homology‐directed repair of a truncated reporter gene (Fauser et al., 2012) and targeted integration via homology‐directed repair into an endogenous locus (Schiml et al., 2014).

The advantage of this approach to targeted transgene integration is that every cell contains donor DNA and nuclease‐encoding sequences such that large numbers of potential homology‐directed targeting events can occur during plant development. Of course, as targeting takes place in somatic tissue, the challenge becomes the identification and proliferation of rare targeted cells, so as to capture the events for germ‐line transmission (Petolino, 2015). In vitro selection for targeting using repair of a selectable marker gene at the target locus was used to effectively isolate targeted events in maize following nuclease‐mediated intragenomic recombination (Ayar et al., 2013).

From a transgene deployment perspective, nuclease‐mediated, homology‐directed, intragenomic recombination has the advantage of avoiding the need to produce large numbers of independent transgenic events in order to generate one that displays targeted integration into a specific locus. As the population of cells containing the editing reagents is generated via crossing plants with stably integrated donor, target and nuclease‐encoding sequences, only a few high‐quality events are required. Moreover, this targeting approach is independent of transformation method. It is particularly powerful with respect to trait stacking whereas new transgenes, flanked by nuclease cleavage sites and DNA sequences homologous to a pre‐integrated target, can be randomly integrated then targeted via intragenomic recombination by crossing with a nuclease‐expressing plant that facilitates cleavage at the target locus, donor excision and homology‐directed repair at the target locus. It is anticipated that nuclease expression in reproductive cells would increase the frequency of targeting germ‐line tissue, thereby avoiding the need for in vitro selection (Even‐Faitelson et al., 2011).

Future prospects

As the demands of modern agriculture become even more complex, the need for genetically enhanced crops with value‐adding performance traits becomes more urgent. The days of single transgenes to impart resistance to single insect pests or tolerance to single herbicides are over. It is anticipated that the future will see broad‐spectrum insect and weed control and performance traits, including stress tolerance and nutrient‐use efficiency, involving multigene stacks. Technologies that increase the precision of genome modification, such as designed nucleases, will play a central role in the development and deployment of such traits. Progress in genome editing using various types of designed nucleases has been rapid in recent years (Gaj et al., 2013). For example, designed nuclease expression, double‐strand break formation at endogenous loci and error‐prone DNA repair has become a routine means of generating targeted mutations (Belhaj et al., 2013). However, there is still a paucity of reports describing the successful integration of incoming donor DNA into predetermined genomic locations (Ainley et al., 2013; Cai et al., 2009; D'Halluin et al., 2008, 2013; Kumar et al., 2015; Shukla et al., 2009; Wright et al., 2005). Moreover, to date, with current technology, the size of transgenic constructs targeted to specific genomic loci in plants has been modest, for example <5 kb. Integrating large DNA sequences into ‘safe harbours’ to minimize position effect and potential unintended consequences, stacking new transgenes into pre‐existing trait loci to simplify segregation and trait introgression and deleting specific sequences to remove or replace transgenes will become more and more routine as gene targeting technology improves. Indeed, the use of homology‐directed, intragenomic recombination, involving the intermating of donor, target and nuclease‐expressing plants, shows great promise for making this technology a reality—even in those crops without a robust transformation capability. This, combined with the use of modular systems involving nuclease‐mediated selectable marker exchange, will provide flexible and generally applicable means of making new trait combinations. The future of agriculture is certainly bright thanks to continued developments in crop improvement technologies such as the designed nuclease.

Acknowledgement

The multitrait product development concepts expressed in this review evolved through discussions with several Dow AgroSciences colleagues including Mike Ainley, Steve Webb, Lakshmi Sastry‐Dent, Manju Gupta and Stephen Novak.

Note

1

‘SartStax® multi‐event technology developed by Dow AgroSciences and Monsanto. SmartStax® is a trademark of Monsanto Technology LLC.’

References

  1. Ainley, W.M. , Sastry‐Dent, L. , Welter, M.E. , Murray, M.G. , Zeitler, B. , Amora, R. , Corbin, D.R. , Miles, R.R. , Arnold, N.L. , Strange, T.L. , Simpson, M.A. , Cao, Z. , Carroll, C. , Pawelczak, K.S. , Blue, R. , West, K. , Rowland, L.M. , Perkins, D. , Samuel, P. , Dewes, C.M. , Shen, L. , Sriram, S. , Evans, S.L. , Rebar, E.J. , Zhang, L. , Gregory, P.D. , Urnov, F.D. , Webb, S.R. and Petolino, J.F. (2013) Trait stacking via targeted genome editing. Plant Biotechnol. J. 11, 1126–1134. [DOI] [PubMed] [Google Scholar]
  2. Alberts, B. and Sternglanz, R. (1990) Gene expression. Chromatin contract to silence. Nature 344, 193–194. [DOI] [PubMed] [Google Scholar]
  3. Antunes, M. , Smith, J.J. , Jantz, D. and Medford, J. (2012) Targeted DNA excision in Arabidopsis by a re‐engineered homing endonuclease. BMC Biotechnol. 12, 86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ayar, A. , Wehrkamp‐Richter, S. , Laffaire, J.B. , Le Goff, S. , Levy, J. , Chaignon, S. , Salmi, H. , Lepicard, A. , Sallaud, C. , Gallego, M.E. , White, C.I. and Paul, W. (2013) Gene targeting in maize by somatic ectopic recombination. Plant Biotechnol. J. 11, 305–314. [DOI] [PubMed] [Google Scholar]
  5. Bejerano, G. , Pheasant, M. , Makunin, I. , Stephen, S. , Kent, W.J. , Mattick, J.S. and Haussler, D. (2004) Ultraconserved elements in the human genome. Science 304, 1321–1325. [DOI] [PubMed] [Google Scholar]
  6. Belhaj, K. , Chaparro‐Garcia, A. , Kamoun, S. and Nekrasov, V. (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9, 39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cai, C.Q. , Doyon, Y. , Ainley, W.M. , Miller, J.C. , Dekelver, R.C. , Moehle, E.A. , Rock, J.M. , Lee, Y.L. , Garrison, R. , Schulenberg, L. , Blue, R. , Worden, A. , Baker, L. , Faraji, F. , Zhang, L. , Holmes, M.C. , Rebar, E.J. , Collingwood, T.N. , Rubin‐Wilson, B. , Gregory, P.D. , Urnov, F.D. and Petolino, J.F. (2009) Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol. Biol. 69, 699–709. [DOI] [PubMed] [Google Scholar]
  8. Chawla, R. , Ariza‐Nieto, M. , Wilson, A.J. , Moore, S.K. and Srivastava, V. (2006) Transgene expression produced by biolistic‐mediated, site‐specific gene integration is consistently inherited by the subsequent generations. Plant Biotechnol. J. 4, 209–218. [DOI] [PubMed] [Google Scholar]
  9. Christian, M. , Cermak, T. , Doyle, E.L. , Schmidt, C. , Zhang, F. , Hummel, A. , Bogdanove, A.J. and Voytas, D.F. (2010) Targeting DNA double‐strand breaks with TAL effector nucleases. Genetics 186, 757–761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clive, J. (2014) Global status of commercialized biotech/GM crops: 2013. Acta Sci. Pol., Hortorum Cultus, 13, 13. [Google Scholar]
  11. Collard, B.C.Y. and Mackill, D.J. (2008) Marker‐assisted selection: an approach for precision plant breeding in the twenty‐first century. Philos. Trans. R. Soc. B: Biol. Sci. 363, 557–572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conner, A.J. and Jacobs, J.M.E. (1999) Genetic engineering of crops as potential source of genetic hazard in the human diet. Mutat. Res. 443, 223–234. [DOI] [PubMed] [Google Scholar]
  13. Crow, J.F. (1998) 90 years ago: the beginning of hybrid maize. Genetics 148, 923–928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dafny‐Yelin, M. and Tzfira, T. (2007) Delivery of multiple transgenes to plant cells. Plant Physiol. 145, 1118–1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dale, E.C. and Ow, D.W. (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc. Natl Acad. Sci. USA 88, 10558–10562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Day, C.D. , Lee, E. , Kobayashi, J. , Holappa, L.D. , Albert, H. and Ow, D.W. (2000) Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles that are differentially silenced. Genes Dev. 14, 2869–2880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dean, C. , Jones, J. , Favreau, M. , Dunsmuir, P. and Bedbrook, J. (1988) Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucleic Acids Res. 16, 9267–9283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Debergh, P.C. and Read, P.E. (1991) Micropropagation. In Micropropagation ( Debergh, P.C. and Zimmerman, R.H. , eds), pp. 1–13. Netherlands: Springer. [Google Scholar]
  19. D'Halluin, K. , Vanderstraeten, C. , Stals, E. , Cornelissen, M. and Ruiter, R. (2008) Homologous recombination: a basis for targeted genome optimization in crop species such as maize. Plant Biotechnol. J. 6, 93–102. [DOI] [PubMed] [Google Scholar]
  20. D'Halluin, K. , Vanderstraeten, C. , Van Hulle, J. , Rosolowska, J. , Van Den Brande, I. , Pennewaert, A. , D'Hont, K. , Bossut, M. , Jantz, D. , Ruiter, R. and Broadhvest, J. (2013) Targeted molecular trait stacking in cotton through targeted double‐strand break induction. Plant Biotechnol. J. 11, 933–941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dockter, C. and Hansson, M. (2015) Improving barley culm robustness for secured crop yield in a changing climate. J. Exp. Bot. 66, 3499–3509. [DOI] [PubMed] [Google Scholar]
  22. Edwards, D. and Batley, J. (2010) Plant genome sequencing: applications for crop improvement. Plant Biotechnol. J. 8, 2–9. [DOI] [PubMed] [Google Scholar]
  23. Epinat, J.C. , Arnould, S. , Chames, P. , Rochaix, P. , Desfontaines, D. , Puzin, C. , Patin, A. , Zanghellini, A. , Paques, F. and Lacroix, E. (2003) A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res. 31, 2952–2962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Even‐Faitelson, L. , Samach, A. , Melamed‐Bessudo, C. , Avivi‐Ragolsky, N. and Levy, A.A. (2011) Localized egg‐cell expression of effector proteins for targeted modification of the Arabidopsis genome . Plant J. 68, 929–937. [DOI] [PubMed] [Google Scholar]
  25. Eyquem, J. , Poirot, L. , Galetto, R. , Scharenberg, A.M. and Smith, J. (2013) Characterization of three loci for homologous gene targeting and transgene expression. Biotechnol. Bioeng. 110, 2225–2235. [DOI] [PubMed] [Google Scholar]
  26. FAO, U. (2009) How to feed the world in 2050. In: Rome: High‐Level Expert Forum, World Summit on Food Security, 16–18.
  27. Fauser, F. , Roth, N. , Pacher, M. , Ilg, G. , Sanchez‐Fernandez, R. , Biesgen, C. and Puchta, H. (2012) In planta gene targeting. Proc. Natl Acad. Sci. USA 109, 7535–7540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ferrie, A.M.R. and Caswell, K.L. (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell, Tissue Organ Cult. 104, 301–309. [Google Scholar]
  29. Ficklin, S.P. , Luo, F. and Feltus, F.A. (2010) The association of multiple interacting genes with specific phenotypes in rice using gene coexpression networks. Plant Physiol. 154, 13–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Gaj, T. , Gersbach, C.A. and Barbas Iii, C.F. (2013) ZFN, TALEN, and CRISPR/Cas‐based methods for genome engineering. Trends Biotechnol. 31, 397–405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Gentry, M. and Hennig, L. (2014) Remodelling chromatin to shape development of plants. Exp. Cell Res. 321, 40–46. [DOI] [PubMed] [Google Scholar]
  32. Gould, F. (1998) Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annu. Rev. Entomol. 43, 701–726. [DOI] [PubMed] [Google Scholar]
  33. Halpin, C. (2005) Gene stacking in transgenic plants–the challenge for 21st century plant biotechnology. Plant Biotechnol. J. 3, 141–155. [DOI] [PubMed] [Google Scholar]
  34. Hare, P.D. and Chua, N.H. (2002) Excision of selectable marker genes from transgenic plants. Nat. Biotechnol. 20, 575–580. [DOI] [PubMed] [Google Scholar]
  35. Hohn, B. , Levy, A.A. and Puchta, H. (2001) Elimination of selection markers from transgenic plants. Curr. Opin. Biotechnol. 12, 139–143. [DOI] [PubMed] [Google Scholar]
  36. Iglesias, V.A. , Moscone, E.A. , Papp, I. , Neuhuber, F. , Michalowski, S. , Phelan, T. , Spiker, S. , Matzke, M. and Matzke, A.J. (1997) Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco. Plant Cell 9, 1251–1264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kim, Y.G. , Cha, J. and Chandrasegaran, S. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Koziel, M.G. , Beland, G.L. , Bowman, C. , Carozzi, N.B. , Crenshaw, R. , Crossland, L. , Dawson, J. , Desai, N. , Hill, M. , Kadwell, S. , Launis, K. , Lewis, K. , Maddox, D. , McPherson, K. , Meghji, M.R. , Merlin, E. , Rhodes, R. , Warren, G.W. , Wright, M. and Evola, S.V. (1993) Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis . Nat. Biotechnol. 11, 194–200. [Google Scholar]
  39. Kumar, S. , AlAbed, D. , Worden, A. , Novak, S. , Wu, H. , Ausmus, C. , Beck, M. , Robinson, H. , Minnicks, T. , Hemingway, D. , Lee, R. , Skaggs, N. , Wang, L. , Marri, P. and Gupta, M. (2015) A modular gene targeting system for sequential transgene stacking in plants. J. Biotechnol. 207, 12–20. [DOI] [PubMed] [Google Scholar]
  40. Kumpatla, S.P. , Chandrasekharan, M.B. , Iyer, L.M. , Guofu, L. and Hall, T.C. (1998) Genome intruder scanning and modulation systems and transgene silencing. Trends Plant Sci. 3, 97–104. [Google Scholar]
  41. van Leeuwen, W. , Ruttink, T. , Borst‐Vrenssen, A.W.M. , van der Plas, L.H.W. and van der Krol, A.R. (2001) Characterization of position‐induced spatial and temporal regulation of transgene promoter activity in plants. J. Exp. Bot. 52, 949–959. [DOI] [PubMed] [Google Scholar]
  42. Lindsey, K. , Wei, W. , Clarke, M. , McArdle, H. , Rooke, L. and Topping, J. (1993) Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants. Transgenic Res. 2, 33–47. [DOI] [PubMed] [Google Scholar]
  43. Lusser, M. , Parisi, C. , Plan, D. and Rodriguez‐Cerezo, E. (2012) Deployment of new biotechnologies in plant breeding. Nat. Biotechnol. 30, 231–239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Marra, M.C. , Piggott, N.E. and Goodwin, B.K. (2010) The anticipated value of SmartStax™ for US corn growers. AgBioForum 13, 1–12. [Google Scholar]
  45. Matzke, A.J. and Matzke, M.A. (1998) Position effects and epigenetic silencing of plant transgenes. Curr. Opin. Plant Biol. 1, 142–148. [DOI] [PubMed] [Google Scholar]
  46. Mlynarova, L. , Keizer, L. , Stiekema, W.J. and Nap, J.P. (1996) Approaching the lower limits of transgene variability. Plant Cell 8, 1589–1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Moehle, E.A. , Rock, J.M. , Lee, Y.L. , Jouvenot, Y. , DeKelver, R.C. , Gregory, P.D. , Urnov, F.D. and Holmes, M.C. (2007) Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl Acad. Sci. USA 104, 3055–3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Moon, H.S. , Li, Y. and Stewart, C.N. Jr (2010) Keeping the genie in the bottle: transgene biocontainment by excision in pollen. Trends Biotechnol. 28, 3–8. [DOI] [PubMed] [Google Scholar]
  49. Mumm, R.H. and Walters, D.S. (2001) Quality control in the development of transgenic crop seed products. Crop Sci. 41, 1381–1389. [Google Scholar]
  50. Odell, J. , Caimi, P. , Sauer, B. and Russell, S. (1990) Site‐directed recombination in the genome of transgenic tobacco. Mol. Gen. Genet. 223, 369–378. [DOI] [PubMed] [Google Scholar]
  51. Padgette, S.R. , Kolacz, K.H. , Delannay, X. , Re, D.B. , LaVallee, B.J. , Tinius, C.N. , Rhodes, W.K. , Otero, Y.I. , Barry, G.F. , Eichholtz, D.A. , Peschke, V.M. , Nida, D.L. , Taylor, N.B. and Kishore, G.M. (1995) Development, identification, and characterization of a glyphosate‐tolerant soybean line. Crop Sci. 35, 1451–1461. [Google Scholar]
  52. Papapetrou, E.P. , Lee, G. , Malani, N. , Setty, M. , Riviere, I. , Tirunagari, L.M. , Kadota, K. , Roth, S.L. , Giardina, P. , Viale, A. , Leslie, C. , Bushman, F.D. , Studer, L. and Sadelain, M. (2011) Genomic safe harbors permit high beta‐globin transgene expression in thalassemia induced pluripotent stem cells. Nat. Biotechnol. 29, 73–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Peleman, J.D. and van der Voort, J.R. (2003) The challenges in marker assisted breeding. In: Eucarpia Leafy Vegetables. ( van Hintum, Th. J. L. , Lebeda, A. , Pink, D. and Schut, J.W. , eds), pp. 125–130. Norwell, MA: : Kluwer Academic Publishers. [Google Scholar]
  54. Petolino, J.F. (2015) Genome editing in plants via designed zinc finger nucleases. In Vitro Cell. Dev. Biol. Plant 51, 1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Petolino, J.F. , Worden, A. , Curlee, K. , Connell, J. , Strange Moynahan, T.L. , Larsen, C. and Russell, S. (2010) Zinc finger nuclease‐mediated transgene deletion. Plant Mol. Biol. 73, 617–628. [DOI] [PubMed] [Google Scholar]
  56. Petolino, J.F. , Sastry‐Dent, L. and Samuel, J.P. (2015) Zinc finger nuclease‐mediated gene targeting in plants. In: Recent Advancements in Gene Expression and Enabling Technologies in Crop Plants ( Azhakanandam, K. , Silverstone, A. , Daniell, H. and Davey, M.R. , eds), pp. 363–381. New York: Springer New York. [Google Scholar]
  57. Puchta, H. , Dujon, B. and Hohn, B. (1996) Two different but related mechanisms are used in plants for the repair of genomic double‐strand breaks by homologous recombination. Proc. Natl Acad. Sci. USA 93, 5055–5060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Qi, Y. , Li, X. , Zhang, Y. , Starker, C.G. , Baltes, N.J. , Zhang, F. , Sander, J.D. , Reyon, D. , Joung, J.K. and Voytas, D.F. (2013) Targeted deletion and inversion of tandemly arrayed genes in Arabidopsis thaliana using zinc finger nucleases. G3: Genes ‐ Genomes ‐ Genetics 3, 1707–1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Que, Q. , Chilton, M.D. , de Fontes, C.M. , He, C. , Nuccio, M. , Zhu, T. , Wu, Y. , Chen, J.S. and Shi, L. (2010) Trait stacking in transgenic crops: challenges and opportunities. GM Crops 1, 220–229. [DOI] [PubMed] [Google Scholar]
  60. Raymond Park, J. , McFarlane, I. , Hartley Phipps, R. and Ceddia, G. (2011) The role of transgenic crops in sustainable development. Plant Biotechnol. J. 9, 2–21. [DOI] [PubMed] [Google Scholar]
  61. Rong, Y.S. and Golic, K.G. (2000) Gene targeting by homologous recombination in Drosophila . Science 288, 2013–2018. [DOI] [PubMed] [Google Scholar]
  62. Schiml, S. , Fauser, F. and Puchta, H. (2014) The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 80, 1139–1150. [DOI] [PubMed] [Google Scholar]
  63. Sharma, D.R. , Kaur, R. and Kumar, K. (1996) Embryo rescue in plants—a review. Euphytica 89, 325–337. [Google Scholar]
  64. Shimatani, Z. , Nishizawa‐Yokoi, A. , Endo, M. , Toki, S. and Terada, R. (2014) Positive–negative‐selection‐mediated gene targeting in rice. Front. Plant Sci. 5, 748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Shukla, V.K. , Doyon, Y. , Miller, J.C. , DeKelver, R.C. , Moehle, E.A. , Worden, S.E. , Mitchell, J.C. , Arnold, N.L. , Gopalan, S. , Meng, X. , Choi, V.M. , Rock, J.M. , Wu, Y.Y. , Katibah, G.E. , Zhifang, G. , McCaskill, D. , Simpson, M.A. , Blakeslee, B. , Greenwalt, S.A. , Butler, H.J. , Hinkley, S.J. , Zhang, L. , Rebar, E.J. , Gregory, P.D. and Urnov, F.D. (2009) Precise genome modification in the crop species Zea mays using zinc‐finger nucleases. Nature 459, 437–441. [DOI] [PubMed] [Google Scholar]
  66. Siebert, M.W. , Nolting, S.P. , Hendrix, W. , Dhavala, S. , Craig, C. , Leonard, B.R. , Stewart, S.D. , All, J. , Musser, F.R. , Buntin, G.D. and Samuel, L. (2012) Evaluation of corn hybrids expressing Cry1F, Cry1A.105, Cry2Ab2, Cry34Ab1/Cry35Ab1, and Cry3Bb1 against southern United States insect pests. J. Econ. Entomol. 105, 1825–1834. [DOI] [PubMed] [Google Scholar]
  67. Sigurbjornsson, B. (1971) Induced mutations in plants. Sci. Am. 224, 86–95. [DOI] [PubMed] [Google Scholar]
  68. Storer, N.P. , Thompson, G.D. and Head, G.P. (2012) Application of pyramided traits against Lepidoptera in insect resistance management for Bt crops. GM Crops Food 3, 154–162. [DOI] [PubMed] [Google Scholar]
  69. Takano, M. , Egawa, H. , Ikeda, J.‐E. and Wakasa, K. (1997) The structures of integration sites in transgenic rice. Plant J. 11, 353–361. [DOI] [PubMed] [Google Scholar]
  70. Wiedenheft, B. , Sternberg, S.H. and Doudna, J.A. (2012) RNA‐guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338. [DOI] [PubMed] [Google Scholar]
  71. Wilson, C. , Bellen, H.J. and Gehring, W.J. (1990) Position effects on eukaryotic gene expression. Annu. Rev. Cell Biol. 6, 679–714. [DOI] [PubMed] [Google Scholar]
  72. Wright, D.A. , Townsend, J.A. , Winfrey, R.J. Jr , Irwin, P.A. , Rajagopal, J. , Lonosky, P.M. , Hall, B.D. , Jondle, M.D. and Voytas, D.F. (2005) High‐frequency homologous recombination in plants mediated by zinc‐finger nucleases. Plant J. 44, 693–705. [DOI] [PubMed] [Google Scholar]
  73. Zhang, W. , Subbarao, S. , Addae, P. , Shen, A. , Armstrong, C. , Peschke, V. and Gilbertson, L. (2003) Cre/lox‐mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor. Appl. Genet. 107, 1157–1168. [DOI] [PubMed] [Google Scholar]
  74. Zhou, H. , Liu, B. , Weeks, D.P. , Spalding, M.H. and Yang, B. (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res. 42, 10903–10914. [DOI] [PMC free article] [PubMed] [Google Scholar]

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