Enabling plant synthetic biology through genome engineering
2014; Elsevier BV; Volume: 33; Issue: 2 Linguagem: Inglês
10.1016/j.tibtech.2014.11.008
ISSN0167-9430
AutoresNicholas J. Baltes, Daniel F. Voytas,
Tópico(s)Plant Virus Research Studies
Resumo•Rewriting genomes will play an important role in plant synthetic biology.•Sequence-specific nucleases enable almost any DNA sequence change in plant cells.•The advantages and limitations of current sequence-specific nucleases are discussed.•A comprehensive list of recent plant genome engineering achievements is provided.•Achievements in genome engineering are related to plant synthetic biology projects. Synthetic biology seeks to create new biological systems, including user-designed plants and plant cells. These systems can be employed for a variety of purposes, ranging from producing compounds of industrial or therapeutic value, to reducing crop losses by altering cellular responses to pathogens or climate change. To realize the full potential of plant synthetic biology, techniques are required that provide control over the genetic code – enabling targeted modifications to DNA sequences within living plant cells. Such control is now within reach owing to recent advances in the use of sequence-specific nucleases to precisely engineer genomes. We discuss here the enormous potential provided by genome engineering for plant synthetic biology. Synthetic biology seeks to create new biological systems, including user-designed plants and plant cells. These systems can be employed for a variety of purposes, ranging from producing compounds of industrial or therapeutic value, to reducing crop losses by altering cellular responses to pathogens or climate change. To realize the full potential of plant synthetic biology, techniques are required that provide control over the genetic code – enabling targeted modifications to DNA sequences within living plant cells. Such control is now within reach owing to recent advances in the use of sequence-specific nucleases to precisely engineer genomes. We discuss here the enormous potential provided by genome engineering for plant synthetic biology. Synthetic biology (see Glossary) is often hard to define because it encompasses a broad range of methodologies for manipulating and harnessing living systems. In simplest terms, synthetic biology combines science and engineering to design and construct new biological parts, devices, and systems [1Arkin A. et al.What's in a name?.Nat. Biotechnol. 2009; 27: 1071-1073Google Scholar]. One area of synthetic biology, and the focus of this review, is the generation of user-designed organisms. These organisms are created for a variety of purposes, ranging from producing valuable compounds that are ultimately purified away from the host to improving the response of an organism to the environment by designing genetic circuits that respond better to external cues. To fully practice in this area of synthetic biology one requires control over DNA sequences, from the in silico design and in vitro synthesis of standardized genetic elements to the in vivo manipulation of host DNA and gene expression. There are now a wide variety of tools available for in vivo manipulation of the genetic material, including recombinases, integrases, RNAi technology, and sequence-specific nucleases, the latter being the focus of this review. Extraordinary advances in sequence-specific nuclease technology within the past 5 years have made it possible for most labs, even those with minimal molecular biology expertise, to precisely manipulate plant genomes, including altering DNA sequences and changing patterns of gene expression. We focus here on sequence-specific nucleases and how they have been used to create genetic modifications for synthetic biology projects. We also discuss future roles for these tools in plant synthetic biology, using examples from several projects, including the ongoing C4 rice project, where photosynthesis in rice is to be completely redesigned for higher efficiency [2Von Caemmerer S. et al.The development of C4 rice: current progress and future challenges.Science. 2012; 336: 1671-1672Google Scholar], and the nitrogen-fixing cereals project, where cereals are to be modified to uptake atmospheric nitrogen [3Oldroyd G.E.D. Dixon R. Biotechnological solutions to the nitrogen problem.Curr. Opin. Biotechnol. 2014; 26: 19-24Google Scholar] (Box 1).Box 1A spotlight on two ambitious synthetic biology projectsExamples of plant synthetic biology projects include the nitrogen-fixing cereals project (Figure IA) and the C4 rice project (Figure IB).By engineering cereals to uptake atmospheric nitrogen, there will be a reduced dependency on inorganic fertilizers. There are two possible approaches for modifying cereals to uptake atmospheric nitrogen: transfer the nodulation signaling pathway from legumes to promote root nodule symbiosis with Rhizobium bacteria, or engineer the nitrogenase enzyme to function in plant cells.Engineering the C4 photosynthesis pathway into C3 rice promises to increase yield. One approach to engineering this pathway in rice is to convert the single-cell C3 cycle into a two-celled C4 cycle. In this case the initial carbon fixation is catalyzed within mesophyll cells by phosphoenolpyruvate carboxylase (PEPC) forming the four-carbon oxaloacetate from bicarbonate and PEP. Oxaloacetate is then metabolized into malate, and the four-carbon acid diffuses into the bundle sheath cell. There, the four-carbon acid is decarboxylated to provide increased concentrations of carbon dioxide to RuBisCO, which is confined in bundle sheath cells. Examples of plant synthetic biology projects include the nitrogen-fixing cereals project (Figure IA) and the C4 rice project (Figure IB). By engineering cereals to uptake atmospheric nitrogen, there will be a reduced dependency on inorganic fertilizers. There are two possible approaches for modifying cereals to uptake atmospheric nitrogen: transfer the nodulation signaling pathway from legumes to promote root nodule symbiosis with Rhizobium bacteria, or engineer the nitrogenase enzyme to function in plant cells. Engineering the C4 photosynthesis pathway into C3 rice promises to increase yield. One approach to engineering this pathway in rice is to convert the single-cell C3 cycle into a two-celled C4 cycle. In this case the initial carbon fixation is catalyzed within mesophyll cells by phosphoenolpyruvate carboxylase (PEPC) forming the four-carbon oxaloacetate from bicarbonate and PEP. Oxaloacetate is then metabolized into malate, and the four-carbon acid diffuses into the bundle sheath cell. There, the four-carbon acid is decarboxylated to provide increased concentrations of carbon dioxide to RuBisCO, which is confined in bundle sheath cells. Plants have largely been unexploited for synthetic biology, but they offer great potential. Plants are the most important source of the primary metabolites that feed the world (i.e., proteins, fatty acids, and carbohydrates) and they also produce a diverse array of secondary metabolites of value for medicine and industry. Further, there is a good understanding of plant systems biology, they are sessile, they can fight off pathogens, and they are not subject to the ethical issues that sometimes limit the use of animal cells. Finally, plants use abundant and inexpensive nutrients (carbon dioxide and sunlight) to produce their primary and secondary metabolites, and their total biomass is enormous: approximately 210 billion tons of plant material are produced each year [4Kircher M. The transition to a bio-economy: emerging from the oil age.Biofuels Bioprod. Biorefining. 2012; 6: 369-375Google Scholar]. Approximately 30 years ago the first plants were generated with novel functions, including herbicide tolerances and insect resistances [5Vaeck M. et al.Transgenic plants protected from insect attack.Nature. 1987; 328: 33-37Google Scholar]. These plants were made through transgenesis, in which user-designed DNA was randomly integrated into plant genomes. While this was an important first step in designing plants with novel functions, the past few years have witnessed the emergence of more sophisticated and precise methods for engineering DNA in living cells. When these methods are used to their fullest potential, they can generate any type of modification within plant genomes, ranging from precisely introducing one or more transgenes at a desired locus, to removing unwanted or unnecessary DNA from the host, to accurately controlling expression of host or synthetic genes. Even by focusing on user-designed plants, the breadth of projects that fall under the synthetic biology term is enormous. Examples of such projects include: (i) modifying cereals, including wheat, to fix atmospheric nitrogen, (ii) redesigning metabolic pathways to increase the yield of secondary metabolites or to generate compounds with enhanced properties, (iii) transferring the C4 photosynthesis pathway to rice, (iv) modifying the glycosylation pathway in plants to accommodate production of therapeutic proteins, and (v) introducing synthetic signal transduction systems that respond to external cues [6Antunes M.S. et al.Engineering key components in a synthetic eukaryotic signal transduction pathway.Mol. Syst. Biol. 2009; 5: 270Google Scholar]. A common ground for most synthetic biology projects is the need for standardized genetic parts (e.g., promoters, terminators, genes), and the subsequent need for tools and techniques for modifying plant genomes. One method to efficiently and precisely modify plant genomes involves introducing targeted DNA double-strand breaks (DSBs) at a locus of interest. Normally, DSBs are highly toxic lesions, and to preserve the integrity of their genomes, all living organisms have evolved pathways to repair such breaks. In general, plant cells have two main DNA repair mechanisms: non-homologous end joining and homologous recombination [7Wyman C. Kanaar R. DNA double-strand break repair: all's well that ends well.Annu. Rev. Genet. 2006; 40: 363-383Google Scholar]. As described in greater detail below, repair by either pathway can be exploited to introduce sequence changes within genomes. In the past two decades, significant effort has been invested in developing reagents that create targeted DNA DSBs. Currently, researchers have a choice between four classes of sequence-specific nucleases: meganucleases, zinc-finger nucleases, TALENs, and CRISPR/Cas (Box 2). All classes are similar in that they can be customized to bind and cleave a target DNA sequence of interest (approximately 18–40 bp in length). Specificity of 18 bp makes it possible to target a single locus in a complex genome. An 18 bp signature occurs once in 68 billion bp of DNA; the wheat genome, for example, is ∼17 billion bp.Box 2Engineering plant DNA using sequence-specific nucleasesDNA DSBs can be targeted to sequences of interest using sequence-specific nucleases. There are four major classes of sequence-specific nucleases: meganucleases, zinc-finger nucleases, TALENs, and CRISPR/Cas (Figure IA). Although these enzymes are structurally different, all can be engineered to recognize and cleave different DNA sequences.MeganucleasesMeganucleases (also referred to as homing endonucleases) were initially found to be encoded by mobile introns, and, since then, they have been repurposed for creating targeted DSBs within genomes. Their relatively small size (∼165 aa) and large DNA recognition sequence (∼18 bp) has made meganucleases an attractive option for genome engineering.Zinc-finger nucleasesZinc-finger nucleases are chimeric fusion proteins that consist of a DNA-binding domain and a DNA-cleavage domain. The DNA-binding domain is composed of a set of Cys2His2 zinc fingers (usually 3–6). Each zinc finger contacts typically 3 bp of DNA, and arrays of 3 or 6 fingers recognize 9 or 18 bp, respectively. The DNA-cleavage domain is derived from the FokI restriction enzyme. FokI activity requires dimerization; therefore, to site-specifically cleave DNA, two zinc-finger nucleases are designed to bind to DNA in a tail-to-tail orientation [58Kim Y.G. et al.Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 1156-1160Google Scholar]. With their relatively small size (∼300 aa per zinc-finger nuclease monomer), and the further advancements in methods for redirecting targeting [14Sander J.D. et al.Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA).Nat. Methods. 2011; 8: 67-69Google Scholar], zinc-finger nucleases should continue to be an effective technology for editing plant DNA.TALENsTranscription activator-like effector nucleases (TALENs) are another class of sequence-specific nucleases, and like zinc-finger nucleases, are composed of a DNA binding domain and a FokI cleavage domain. The DNA binding domain is derived from TALE proteins found in Xanthamonas sp. Each TALE DNA binding domain is composed of repeat sequences consisting of 33–35 aa. Within each repeat are two variable amino acids (RVDs) that facilitate binding to a single DNA base.CRISPR/Cas and alternative CRISPR/Cas nucleasesThe CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas (CRISPR-associated) system is the most recent addition to the family of sequence-specific nucleases. The CRISPR/Cas system employed for genome engineering consists of a Cas9 endonuclease and a guide RNA (gRNA). Approximately 20 nucleotides within the gRNA are responsible for directing Cas9 cleavage. A protospacer adjacent motif (PAM) is required for DNA cleavage. To increase target specificity, DSBs can be generated using dCas9 (nuclease-inactive Cas9) fusions to FokI, or using Cas9 nickases (Figure IB). DNA DSBs can be targeted to sequences of interest using sequence-specific nucleases. There are four major classes of sequence-specific nucleases: meganucleases, zinc-finger nucleases, TALENs, and CRISPR/Cas (Figure IA). Although these enzymes are structurally different, all can be engineered to recognize and cleave different DNA sequences. Meganucleases Meganucleases (also referred to as homing endonucleases) were initially found to be encoded by mobile introns, and, since then, they have been repurposed for creating targeted DSBs within genomes. Their relatively small size (∼165 aa) and large DNA recognition sequence (∼18 bp) has made meganucleases an attractive option for genome engineering. Zinc-finger nucleases Zinc-finger nucleases are chimeric fusion proteins that consist of a DNA-binding domain and a DNA-cleavage domain. The DNA-binding domain is composed of a set of Cys2His2 zinc fingers (usually 3–6). Each zinc finger contacts typically 3 bp of DNA, and arrays of 3 or 6 fingers recognize 9 or 18 bp, respectively. The DNA-cleavage domain is derived from the FokI restriction enzyme. FokI activity requires dimerization; therefore, to site-specifically cleave DNA, two zinc-finger nucleases are designed to bind to DNA in a tail-to-tail orientation [58Kim Y.G. et al.Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 1156-1160Google Scholar]. With their relatively small size (∼300 aa per zinc-finger nuclease monomer), and the further advancements in methods for redirecting targeting [14Sander J.D. et al.Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA).Nat. Methods. 2011; 8: 67-69Google Scholar], zinc-finger nucleases should continue to be an effective technology for editing plant DNA. TALENs Transcription activator-like effector nucleases (TALENs) are another class of sequence-specific nucleases, and like zinc-finger nucleases, are composed of a DNA binding domain and a FokI cleavage domain. The DNA binding domain is derived from TALE proteins found in Xanthamonas sp. Each TALE DNA binding domain is composed of repeat sequences consisting of 33–35 aa. Within each repeat are two variable amino acids (RVDs) that facilitate binding to a single DNA base. CRISPR/Cas and alternative CRISPR/Cas nucleases The CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas (CRISPR-associated) system is the most recent addition to the family of sequence-specific nucleases. The CRISPR/Cas system employed for genome engineering consists of a Cas9 endonuclease and a guide RNA (gRNA). Approximately 20 nucleotides within the gRNA are responsible for directing Cas9 cleavage. A protospacer adjacent motif (PAM) is required for DNA cleavage. To increase target specificity, DSBs can be generated using dCas9 (nuclease-inactive Cas9) fusions to FokI, or using Cas9 nickases (Figure IB). One of the first steps in engineering plant genomes is to design and construct one or multiple sequence-specific nucleases. How does one choose between the different classes of nucleases? We list here the defining characteristics of each class to help researchers make informed decisions about nuclease choice. Meganucleases were the first class of sequence-specific nucleases used in plants [8Puchta H. et al.Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5055-5060Google Scholar], and they continue to be deployed to achieve complex genome modifications [9D’Halluin K. et al.Targeted molecular trait stacking in cotton through targeted double-strand break induction.Plant Biotechnol. J. 2013; 11: 933-941Google Scholar]. An advantage of meganucleases is their size. They are among the smallest nucleases – comprising only ∼165 amino acids (aa) – making them amenable to most delivery methods, including vectors with limited cargo capacities, such as plant RNA viruses [10Marton I. et al.Nontransgenic genome modification in plant cells.Plant Physiol. 2010; 154: 1079-1087Google Scholar]. Relative to other sequence-specific nucleases, however, meganucleases are challenging to redesign for new target specificity. Redesign is hindered by the non-modular nature of the protein. For example, within the LAGLIDADG family of meganucleases, the amino acids responsible for binding DNA overlap with those for DNA cleavage [11Prieto J. et al.The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage.Nucleic Acids Res. 2007; 35: 3262-3271Google Scholar]; therefore, attempting to alter the DNA-binding domain can affect the enzyme's catalytic activity. As a result, the use of meganucleases in plants has been limited to naturally occurring meganucleases (e.g., I-SceI, I-CreI) or to redesigned nucleases made by groups with expertise in structure-based design or the capacity to carry out high-throughput in vitro screens to identify active nucleases from libraries of variants. Like the meganucleases, zinc-finger nucleases are relatively small (∼300 aa per monomer; ∼600 aa per nuclease pair), making them amenable to most delivery methods. DNA targeting by zinc-finger nucleases is achieved by arrays of zinc fingers, each of which typically binds to a nucleotide triplet. Whereas redesigning the zinc-finger DNA-binding domain is not as difficult as for meganucleases, there are still challenges in achieving new target specificity, mostly due to the influence of context on zinc-finger function. For example, a zinc finger that recognizes GGG in one array may not recognize this sequence when positioned next to different zinc fingers. As a result, modular assembly of zinc fingers has had limited success [12Ramirez C.L. et al.Unexpected failure rates for modular assembly of engineered zinc fingers.Nat. Methods. 2008; 5: 374-375Google Scholar]. One of the more successful methods for redirecting targeting involves screening libraries of three zinc-finger variants to identify those that best recognize and bind to their intended target sequence [13Maeder M.L. et al.Rapid ‘open-source’ engineering of customized zinc-finger nucleases for highly efficient gene modification.Mol. Cell. 2008; 31: 294-301Google Scholar]. More recently, modular methods for constructing zinc-finger arrays have been successful that use two-finger units to minimize context effects [14Sander J.D. et al.Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA).Nat. Methods. 2011; 8: 67-69Google Scholar]. Consequently, generating functional zinc-finger nucleases is now achievable by most research labs. TALENs are a recent addition to the arsenal of sequence-specific nucleases, and they quickly became adopted for plant genome engineering. One advantage of TALENs, compared to meganucleases and zinc-finger nucleases, is their modular DNA binding domain. The TALE DNA binding domain is composed of direct repeats consisting of 33–35 aa. Two amino acids within these repeats, termed repeat-variable diresidues (RVDs), recognize a target nucleotide (e.g., the most widely used RVDs and their nucleotide targets are HD, cytosine; NG, thymine; NI, adenine; and NN, guanine and adenine). This one-to-one correspondence of a single RVD to a single DNA base, together with effective methods for cloning arrays of the DNA binding motif [15Cermak T. et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39: e82Google Scholar, 16Reyon D. et al.FLASH assembly of TALENs for high-throughput genome editing.Nat. Biotechnol. 2012; 30: 460-465Google Scholar, 17Briggs A.W. et al.Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers.Nucleic Acids Res. 2012; 40: e117Google Scholar, 18Schmid-Burgk J.L. et al.A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes.Nat. Biotechnol. 2013; 31: 76-81Google Scholar], have nearly eliminated the design challenges encountered with zinc-finger nucleases and meganucleases. Another advantage of TALENs is their target specificity. TALEN monomers are typically designed with 15–20 RVDs, and, as a result, a TALEN target site is frequently >30 bp. This relatively large target site makes TALENs the most specific of all the nucleases, and may contribute to reduced toxicity compared to zinc-finger nucleases [19Mussolino C. et al.TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity.Nucleic Acids Res. 2014; 42: 6762-6773Google Scholar]. The only real drawback to the use of TALENs is their large size (∼950 aa; ∼1900 aa per pair) and repetitive nature, making delivery to plant cells a challenge. TALENs are typically delivered to plant cells by direct delivery of DNA to protoplasts, or by stable integration of TALEN-encoding constructs into plant genomes. The most recent addition to the sequence-specific nuclease family, CRISPR/Cas, is proving to be the nuclease-of-choice for plant genome engineering. Unlike the other three nuclease classes, which target DNA through protein/DNA interactions, CRISPR/Cas uses a guide RNA molecule (gRNA) to direct an endonuclease, Cas9, to a target DNA sequence. As a result, redirecting CRISPR/Cas is extremely simple, requiring only the cloning of a 20 nt sequence (complementary to a target DNA sequence) within a gRNA expression construct. One limitation of the CRISPR/Cas system may be off-target cleavage [20Fu Y. et al.High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells.Nat. Biotechnol. 2013; 31: 822-826Google Scholar, 21Cho S.W. et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141Google Scholar]. Whereas 20 nucleotides are used to direct Cas9 binding and cleavage, the system tolerates mismatches, with a higher tolerance for mismatches at the 5′ end of the targeting-RNA sequence [20Fu Y. et al.High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells.Nat. Biotechnol. 2013; 31: 822-826Google Scholar]. To reduce the likelihood of off-target cleavage, alternative CRISPR/Cas reagents have been developed, including paired Cas9 nickases [22Ran F.A. et al.Double nicking by RNA-guided CRISPR cas9 for enhanced genome editing specificity.Cell. 2013; 154: 1380-1389Google Scholar, 23Mali P. et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nat. Biotechnol. 2013; 31: 833-838Google Scholar], fusion of catalytically-dead Cas9 to FokI [24Guilinger J.P. et al.Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.Nat. Biotechnol. 2014; 32: 577-582Google Scholar, 25Tsai S.Q. et al.Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.Nat. Biotechnol. 2014; 32: 569-576Google Scholar], and shortening of the gRNA targeting sequence [26Fu Y. et al.Improving CRISPR–Cas nuclease specificity using truncated guide RNAs.Nat. Biotechnol. 2014; 32: 279-284Google Scholar]. Possibly the simplest approach to minimize off-target cleavage is to design gRNAs that have minimum sequence homology to other sites within the plant genome. In addition to potential off-targeting, another limitation of the CRISPR/Cas system is size. Cas9 is ∼1400 aa, making it one of the largest sequence-specific nucleases. However, for vectors that are unable to deliver Cas9, it may be possible to generate plant lines that constitutively express Cas9; therefore, only the delivery of gRNA(s) is required. To help describe how genome engineering can contribute to synthetic biology, we categorize the goals of synthetic biology projects into three groups: those that require precise insertion of DNA into plants genomes, those that require elimination or adjustment of host sequence, and those that require control over transcription of host or non-host genes. We describe how these modifications can be achieved using sequence-specific nucleases, and how they relate to synthetic biology projects. Generating plants with novel function frequently requires integrating foreign DNA (e.g., promoters, genes, terminators, and other transcription regulatory elements) into the plant genome. Conventional approaches for delivering this DNA include Agrobacterium or biolistics, both of which result in the random integration of one or more copies of the DNA sequence. While effective, these methods have several limitations: transgene expression often varies depending on chromosomal context and, when multiple transgenes are integrated at random sites on different chromosomes, they segregate independently, presenting a challenge for breeding regimes that seek to move transgenes into new germplasm. Using sequence-specific nucleases, foreign DNA can be precisely integrated at a locus of interest either through homologous recombination or non-homologous end joining (NHEJ)-mediated insertion (Figure 1). Not only does this enable trait stacking to expedite breeding efforts, but it may reduce variability in gene expression. Because of the minimal targeting constraints for TALENs and CRISPR/Cas systems, nearly all chromosomal positions are amenable to site-specific integration. Notably, there may be sites that are inaccessible or difficult to cleave because of epigenetic factors (e.g., chromatin structure, methylation) or genetic factors (e.g., repetitive DNA that permits nuclease binding to multiple sites within a genome). Transgene stacking was first demonstrated in maize at a preintegrated synthetic target sequence [27Ainley W.M. et al.Trait stacking via targeted genome editing.Plant Biotechnol. J. 2013; 11: 1126-1134Google Scholar] (Table 1). In this case the target for integration was a transgene construct that included the herbicide-tolerance gene phosphinothricin acetyltransferase (PAT) followed by a ‘trait landing pad’ with zinc-finger nuclease target sites flanked by DNA sequences for recombining with an incoming donor molecule. After co-transforming immature embryos with DNA encoding the zinc-finger nucleases and donor DNA (containing a second herbicide tolerance gene, AAD1, flanked by sequences of homology to the trait landing pad), 5% of the transgenic events contained the targeted integration event. Using a similar approach, trait stacking was accomplished in cotton; however, instead of using pre-engineered zinc-finger nucleases to break a synthetic target sequence, a meganuclease was designed to cleave an endogenous locus [9D’Halluin K. et al.Targeted molecular trait stacking in cotton through targeted double-strand break induction.Plant Biotechnol. J. 2013; 11: 933-941Google Scholar].Table 1List of genome modifications achieved in plants using sequence-specific nucleasesType of DNA modificationNucleaseDelivery method(s)Donor?Plant(s)Target(s)RefsTrait stackingMeganucleaseBombardmentYesCottonIntergenic sequence9D’Halluin K. et al.Targeted molecular trait stacking in cotton through targeted double-strand break induction.Plant Biotechnol. J. 2013; 11: 933-941Google ScholarZinc-finger nucleaseBombardmentYesZea maysTransgene27Ainley W.M. et al.Trait stacking via targeted genome editing.Plant Biotechnol. J. 2013; 11: 1126-1134Google ScholarRewriting host DNA: gene knockoutMeganucleaseStable integrationNoZea maysIntergenic sequence59Gao H. et al.Heritable targeted mutagenesis in maize using a designed endonuclease.Plant J. 2010; 61: 176-187Google ScholarMeganucleaseStable integration; Agrobacterium T-DNA (transient)NoZea maysMS2660Djukanovic V. et al.Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designed I-CreI homing endonuclease.Plant J. 2013; 76: 888-899Google ScholarZinc-finger nucleaseStable integrationNoArabidopsis thalianaADH1, TT434Zhang F. et al.High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 12028-12033Google ScholarZinc-finger nucleaseStable integrationNoGlycine maxDCL1a/b, DCL4a/b, RDR6a, HEN1a, transgene61Curtin S.J. et al.Targeted mutagenesis of duplicated genes in soybean with zinc-fi
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