Transcription activator‐like effector nucleases (TALENs): An efficient tool for plant genome editing

Abstract

Transcription activator‐like effector nucleases (TALENs) are a novel class of genome editing tools that provide precise insertion, deletion, or substitution of specific genes in order to alter the genome. TALENs consist of a designed TALE domain that mimics the natural transcription activator‐like effector proteins and a nuclease that can cleave DNA in cells. TALENs have recently emerged as a revolutionary genome editing tools in numerous species and cell types. Here, we review the development of TALEN technology and summarize the principles and tools for TALEN‐mediated genome modification in plant cells.

Abbreviations
• aas
• amino acids
• CRISPR
• clustered regularly interspaced palindromic repeats
• cTALEN
• compact TALEN
• DSB
• double-strand break
• FLASH
• fast ligation-based automatable solid-phase high-throughput
• HR
• homologous recombination
• ICA
• iterative capped assembly
• INDEL
• insertion or deletion
• NHEJ
• nonhomologous end joining
• RVD
• repeat variable di-residue
• ZFN
• zinc-finger nuclease

1 Introduction

The development in targeted genome modifications enables researchers to modify gene of interest to investigate its function and explore its biotechnology applications for genetic improvement of plants and many other organisms. In order to execute the requisite and efficient genetic modification, the site-specific DNA double-strand break (DSB) and the consequent DNA repair by homologous recombination (HR) or nonhomologous end joining (NHEJ) is a decisive step. NHEJ is mostly imprecise leading to gene knockout by introducing nonspecific short deletions or insertions. NHEJ uses several repair enzymes to join the two DSB ends by direct ligation. NHEJ repair process can take place via two modes: the Ku-dependent and Ku-independent NHEJ . In Ku-dependent NHEJ, the Ku heterodimer—Ku70/Ku80 proteins binds to the broken DNA ends and brings them together by formation of a synaptic complex. This complex serves as a platform for the recruitment of repair enzyme DNA ligase IV and its cofactor to join both the ends. In certain cases, the Ku-dependent pathway can produce several nucleotide insertions or deletions (INDELs) at the DSB site. Alternatively, the Ku-independent pathway can repair DSBs in eukaryotes. Microhomology-mediated end joining is a major Ku-independent NHEJ pathway and produce longer deletions at the DSB site compared with Ku-dependent NHEJ and commonly leads to chromosome translocations . HR is usually an error-free DSB repair pathway and is the responsive factor for the genetic exchange between the host and foreign DNA fragments, achieved through its flanking homologous sequences. This is mediated by recombinase proteins (Rad51, Rad52, and Dmc1etc.,) which catalyzes homologous DNA pairing and strand exchange resulting in gene insertions, gene deletions, gene replacement, gene doubling, and precise gene editing etc. . HR is restricted to late S or G2 phase cells, on the other hand, NHEJ operates throughout the entire cell cycle. Hence, NHEJ is a major DSB repair pathway in eukaryote. Both NHEJ and HR repair pathways can be applied for nuclease-based genome editing (Fig. ).

Overview of the DNA DSB and the consequent repair mechanism. Actions of TALENs: The conventional TALENs require two corresponding DNA recognition sites (TALEN-F and TALEN-R) flanking an unspecific central spacer region. The dimeric form of the FokI nuclease domain, fused by a single polypeptide, shown in brown line is necessary for DSB. The genomic DSBs generated by TALENs can be repaired by HR or NHEJ resulting in the desired genome editing. The NHEJ-mediated pathway leads to the generation of small INDELs. HR with double-stranded repair template-DNA leads to precise gene editing and gene replacement.

Several different types of synthetic molecules have been attempted to make targeted DSBs from improved understandings of DNA-binding proteins, which act as sequence-specific nucleases, a combination of a DNA recognition part and a nuclease part. The typical sequence-specific nucleases are zinc-finger nucleases (ZFNs), TALENs, Clustered regularly interspaced palindromic repeats (CRISPR/Cas9), and meganucleases. ZFNs have been developed to generate site-specific DSBs for targeted genome modification. ZFNs are synthetic proteins manufactured by fusing various Zinc-Finger modules (finger-like section of certain DNA-binding proteins) to a sequence-independent cleavage domain of restriction enzyme FokI . Each ZF module consists of 30 amino acids (aas) bound to coordinates one zinc atom using two histidine and two cystine residues. The α-helix in each ZF module can recognize a specific endonuclease DNA codon, enabling them to recognize long DNA sequences . The independent DNA binding and cleavage domains can be optimized in isolation for effective DNA cleavage against custom-designed target sites. ZFNs have been applied for precise genome modifications in a wide range of organisms including plants . Transcription activator-like effector nucleases (TALENs) in particular, consisting of a free designable DNA-binding domain and a nuclease, have been explored today by a large number of researchers in many different organisms. Like ZFNs, TALENs use the nonspecific FokI cleavage domain and they function as dimers. However, the customizable DNA-binding domains of TALENs are composed of a series of short tandem repeats as in TALEs of the plant pathogenic bacteria from the genus Xanthomonas . TALEs are natural type III effector proteins secreted by Xanthomonas spp. to modulate gene expression in host plants. TALEs proteins contain a nuclear localization signal, a transcriptional activation domain, and a central DNA-binding domain . The nuclear localization signal allows TALEs to enter inside a plant nucleus and the activation domain activates the transcriptional machinery for gene expression . The central DNA-binding domain of each TALE consists of varying numbers of repeat units, typically 33–35 aa that recognize a single nucleotide. The last repeat is typically shorter, with only 20 aa, and is therefore called a “half-repeat.” These repeat units are highly conserved with the exception of two variable aas at positions 12 and 13 . The residues called the Repeat Variable Di-residues (RVDs) are responsible for the DNA-binding specificity of the repeat region (Fig. ). The number and order of RVD in each repeat determine the nucleotide-binding specificity. The binding of TALEs to DNA sequences is highly specific due to unique combinations of RVDs. More than 20 unique RVD sequences have been identified in TALEs, but only seven are common and according to this, HD recognizes cytosine (C), NG recognizes thymine (T), NI recognizes adenine (A), NK recognizes guanine (G), NN recognizes A,G nucleotide, and NS can bind to A, C, G, or T with equal affinity . In addition, NH has been shown to recognize G with higher specificity than NN .

Design of native TALEN structure, cTALEN, and single-chain TALEN. (A) Native TALE from Xanthomonas is composed of DNA-binding domain, nuclear localization signal (NLS), transcriptional activation domain, and C-terminal domain. The DNA-binding domain is composed of multiple repeats that are identical except for two signature aas at positions 12 and 13. The four colors box indicates modules for each of the four Bp. Functional domains such as repressors, activators, nucleases, and methylases can be used with native TALE to design TALENs for site-specific genome modification. Designed TALENs can recognize the target sites of two TALE-binding sites, which are separated by spacer region. The spacer is recognized by FokI nuclease. (B) cTALEN is composed of native TALE backbone fused with a single TevI homing endonuclease. (C) Single-chain TALEN is composed of a native TALE DNA-binding domain and two FokI nuclease domains, which are connected by a single polypeptide linker.

More recently, the crystal structure of TALE-DNA complex furnished elaborative information for one-to-one RVD-nucleotide recognition code. The study showed that each TAL repeat comprise of two helices connected by a short RVD-containing loop. The repeats are self-associated to form a right-handed super helical structure wrapped along the sense-strand of the DNA duplex, with the RVDs contacting the major groove . The first residue of each RVD (position 12, either His or Asn) forms hydrogen bonds to the backbone carbonyl oxygen of Ala at position 8 (which is located at the C-terminal end of helix) of each repeat. Hence, the primary role of residue 12 is to stabilize the conformation of the RVD loop and not to recognize nucleotide sequence. The second aa (at position 13) determines specificity by interacting directly with the nucleotide via hydrogen bonds or vanderwalls interactions. TALENs have emerged as powerful alternative to ZFNs, since they are simpler and cheaper to design and have been demonstrated to have great potential for targeted genome modification . Therefore, unlike the context-dependent DNA binding of ZFNs, TALENs can be easily engineered to target nearly any DNA sequence because of their simplistic protein-DNA code specificity. Besides, TALENs exhibit significantly reduced off-target effects compared to ZFNs, making them next-generation tool for genome editing.

Very recently, a new DNA targeting platform has been developed from a natural adaptive immune system of bacteria and archaea called CRISPR-associated genes . This system is based on the usage of Cas9 endonuclease and an engineered single-stranded guide RNA. The guide RNAs recruit the Cas9 endonuclease protein to their target DNA sequence, which leads to the cleavage of the target sequence. This, subsequently, results in gene modification through NHEJ and HR mechanisms. Meganucleases are another class of naturally occurring endonucleases found in different organisms such as bacteria, fungi, and algae. They have a long DNA recognition site with variable size differing between 12 and 30 Bp. Naturally occurring meganucleases can be altered by introducing changes to the aas found in the recognition site of the protein to adapt for restriction of a specifically chosen sequence.

2 Designing of TALENs

The selection of the target sites is the primary interest in TALEN designing. In 2011, Bogdanove and Voytas described the criteria for designing of TALEN target sites, which are given below:

• The targeted sequence must start with a T, referred to as T0.

• T should be disfavored at position 1.

• A should be disfavored at position 2.

• The last nucleotide of the target sequence should be T, so that a strong bias will reflect for NG at the last position of the corresponding TALEN (repeat array).

• The target DNA should have a low G content (9 ± 8%).

• Natural TALE has an average nucleotide composition of the binding site of 31 ± 16% A, 37 ± 10% C, 9 ± 8% G, and 22 ± 10% T. Thus, the potential TALEN target site base composition should contain A = 0–63%, C = 11–63%, G = 0–25%, and T = 2–42%.

3 Assembly of TALE repeats

Due to the high repetitive nature of the DNA-binding domains of TALEs, it is ambitious to construct plasmids encoding long arrays of TALE repeats. In order to overcome this limitation, a number of methods have been developed by many laboratories for assembling the highly repetitive TALE central repeats in a quick and cost-effective manner . Most of the methods used Type IIS restriction enzymes, in order to perform restriction and ligation in a single reaction. Depending on their assembly theories, TALENs are constructed by the following methods.

4 Golden Gate cloning-based assembly

Because of its user-friendly approach, simple and fast processing, Golden Gate assembly method has been widely applied in construction of TALENs. It uses Type IIS restriction endonucleases (e.g. BSaI, BsmBI, and BbsI) to cleave outside of their recognition site sequence and produce nonpalindromic, 4bp 5′-overhang . Since, the ligated product of interest does not contain the original Type IIS restriction site and thus will not be subjected to redigestion in a restriction–ligation reaction. Therefore, the cleavage and ligation can be carried out in the same reaction mixture in a single step and up to 10 TALE repeats can be ligated in one reaction. PCR-based Golden Gate is a modified method of Golden Gate, and uses PCR fragments instead of plasmid fragments. About 4–6 TALE repeat units can be ligated in a single reaction employing this method. So far, most of the TALENs targeting plant genes have been made by the Golden Gate technology.

5 High-throughput solid-phase assembly

Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) technology was developed by Reyon et al. to accomplish large-scale assembly of TALEs. DNA fragments encoding TAL effector repeats are assembled in an iterative fashion on solid-phase magnetic beads. In each cycle, additional DNA fragments encoding preassembled TAL effector repeats are ligated to the immobilized DNA fragments until an array of desired length is assembled. FLASH technology builds TALEs from oligomers (mostly 4-mers) rather than monomers, thereby reducing the number of ligation reactions. Once cleaved from the magnetic bead, the tail domain is subsequently cloned into the desired expression vector. The FLASH technology using an automated liquid handling station can assemble DNA fragments encoding up to 96 different TAL effector repeat arrays in less than a day , while a manual process using multichannel pipettes takes 1–2 days.

Iterative Capped Assembly (ICA) is another kind of solid-phase synthesis method of TALENs construction developed by Church and co-workers' laboratory . ICA involves rapid assembly of repeat module DNA by sequential ligation of monomers on a solid support together with capping oligonucleotides. The capping oligonucleotides are used to prevent monomer self-ligation in each step, which are essential for the production of full length TALE repeat array. An advantage of ICA lies in a fact that it does not depend on preassembled RVDs library, but, each individual RVD is added sequentially. Recently, a ligation-independent cloning technique has been developed for high-throughput assembly of TALE genes . They generated a 5-mer TALE repeat fragment library for the synthesis of TALEs. This method has the advantage of high fidelity, high throughput, and automation. These high-throughput TALEN assembly platforms make it convenient to edit the genes on a genome-wide scale.

6 TALENs target site prediction tools

TALENs have now become an accepted tool for targeted mutagenesis. However, undesired off-targets still remain an important issue. In the past few years, various computational algorithms have been developed for identifying potential off-target sites spanning a wide range of approaches and techniques. The search parameters can be fixed by the user to work with any TAL effector or TAL effector nuclease architecture. Some of these have been discussed in Table .

List of some popular computational algorithms available for TALEN target site predication

Name of software	URL	Features	References
TALE-NT	http://boglab.plp.iastate.edu/	TALE-NT is designed to search for TALEN targets in query sequences, with any repeat length and spacer length. Multiple features are provided, such as finding restriction enzyme site in TALEN spacer and e-mail notification.
idTALE	http://idtale.kaust.edu.sa/gene_id.html	idTALE can search TALEN sites in query sequences or genes by inputting IDs of certain species. The length of the TALE repeat units and specified by users, but the left and right half-site must have the same length.
E-TALEN	http://www.e-talen.org.	It is a web-based tool for TALEN design to generate genome modification constructs. It can be design specific and efficient TALENs against single genes or large number of genes. It also checks the possibility of restriction sites in the target sequence or in the spacer region.
Mojo Hand	www.talendesign.org	Mojo Hand appears more flexible; any gene or sequence can be used and restriction analysis is also available.
TALgetter	http://galaxy.informatik.uni-halle.de	TALgetter is a new approach for predication of TALE target sites, preferentially located in the vicinity of the transcription start site.
TALENoffer	http://jstacs.de/index.php/TALENoffer	It is a tool for genome-wide predication of TALEN targets and off-targets.
Talvez	http://bioinfo.mpl.ird.fr/cgi-bin/talvez/talvez.cgi	It is based on Positional Weight Matrix (PWM) method, which scores all possible binding sites in a promoter region.
Storyteller	http://bioinfo.mpl.ird.fr/xantho/tales	Storyteller converts a TALE RVD sequence into a PWM and uses this matrix to generate a set of possible binding sequences and takes the advantage of a faster pattern—search algorithm based on Hidden Markov Models (HMM).

7 Delivery of TALENs into target cells

The breakthrough of TALENs technology has opened up new means for targeted genome modification. However, the delivery of TALEN proteins into target cells poses a major hurdle due to their large size and high repetitive nature of the TALE DNA-binding domain. The high-repetitive TALE sequences are prone to extensive rearrangement if delivered through lentiviral vectors . In addition, the bulky size of the TALENs has limited their delivery into cells because of space-constrained delivery vehicles such as adeno-associated viruses. However, transfection of TALENs-encoded plasmid DNA or mRNA extends an alternate approach to viral-based methods, but these techniques are restricted to specific cell types and may be highly toxic. Recently, Liu et al. published a new method for TALENs delivery by Cell-penetrating peptide. Cell-penetrating peptide can be reversibly conjugated to cys residues present on the surface of TALE domains to bring cell-penetrating activity to TALENs proteins. They demonstrated that R9-conjugated TALENs have induced gene knockout for the human CCR and BMPR1A genes in HeLa and HEK293 cells, respectively, with no overt toxicity.

To simplify vectorization, Beurdeley et al. had designed a compact TALEN (cTALEN) architecture by replacing the dimeric FokI domain with the GIY-YIG homing endonuclease I-TevI to a TALE-binding scaffold, which eventually resulted in a combination of single-chain TALEN and a nickase function. By this design, cTALENs can cleave the targeted DNA sequence as a monomer and thus reduce the TALEN payload to half as compared with the conventional TALEN architecture. cTALENs offer greater flexibility via a reduced vectorization payload with significant activity. Furthermore, Sun and Zhao developed a single-chain TALEN architecture in which they fused two FokI nuclease domains to a single polypeptide linker. These novel designed TALENs architecture reduce the protein payload and serve as a template for efficient genome editing tool.

In plants, Agrobacterium-mediated transformation, PEG-mediated transformation, and biolistic methods have been exploited for TALENs delivery. Furthermore, TALENs can also be expressed transiently by electroporation, chemical method, or agroinfiltration in plant protoplast or leaves, followed by regeneration employing tissue culture methods. But the plant regeneration by these methods have low success rate. Thus, efficient systems to deliver TALENs into plant cells need to be developed to overcome these limitations. Recently, geminivirus-based replicons were used for the delivery of TALENs proteins as well as DNA repair template into cells . This showed a high rate of in vivo gene targeting because its DNA genome served as a repair template and can replicate with high copy number in addition to the pleiotropic activity of geminiviral Replication initiator proteins. Geminivirus can infect monocots and dicots, offer huge potential as a vector for genome editing technologies . The use of viral vectors to deliver TALENs into plant cells has provided several advantages over other delivering agents, which include high level of expression, low toxicity, and nonintegration of TALENs genes into the host genome.

8 Application of TALENs in crop plants

TALENs-mediated gene modification is a versatile and current-generation tool for site-specific modification of plant genome and has potential to frame enormous impact on crop improvement. So far, TALEN-mediated genome modification has been applied in various plants species listed in Table . The first report of crop improvement employing TALENs technology was reported in rice. Xanthomonas oryzae is a causal pathogen for blight disease, leads to significant annual loss in rice production worldwide. During the infection, bacterial effector protein binds to the promoter region of the sucrose-efflux transporter gene (OsSWEET 14), which activates specific rice disease-susceptible genes. A pair of TALENs that targeted the promoter region of the OsSWEET 14 gene was designed and transformed into rice. The stable transformation of these TALENs resulted in mutation in the effector binding site in the promoter region, which in turn, resulted in silencing of the gene, and consequent resistance to X. oryzae . In barley, TALENs were used to target the promoter region of a phytase gene HvPaphya. The stable transformed plants contained a variety of INDELs. Based on a PCR restriction assay, 16–31% of transformed plants contained some type of INDELs . TALENs were utilized to achieve site-directed mutagenesis in soybean. The fatty acid desaturase genes (FAD2-1A and FAD2-1B), which convert the oleic acid into linoleic acid, were selected as targets. The desaturase gene was successfully mutated when plants were stably transformed. The fatty acid profile of the seed of stably mutated plants showed the production of oleic acid to be nearly four times higher compared to the parents .

List of reported targeted gene(s) via TALENs technology in different plant species

Plant species	Genes	TALEN assembly method	Delivery methods	References
Arabidopsis thaliana	ADH1 ADH1, TT4, MAPKKK1, DSK2Ba, DSK2Bb, NATA2a, NATA2b.	Golden Gate	Protoplast transformation; Agrobacterium-mediated floral dip transformation
Brachypodium	BdABA1,BdCKX2,BdMC6, BdSPL,BdHRT,BdSPP,BdHTA1, BdCO11.	Golden Gate	Protoplast and Agrobacterium-mediated transformation
Tobacco	SurA,SurB.	Golden Gate	Protoplast transformation
Rice	OsBADH2,OsDEP1,OsSD1, OsCKX2, Os11N3.	Golden Gate	Protoplast and Agrobacterium-mediated transformation.
Barley	HvPAPhy	Golden Gate	Agrobacterium-mediated transformation
Soybean	FAD2-1A, FAD2-1B.	Golden Gate	Agrobacterium-mediated transformation
Wheat	TaMLO-A1, TaMLO-B1, TaMLO-D1	Golden Gate	Protoplast transformation and Biolistic transformation
Tomato	PROCERA	Golden Gate	Agrobacterium-mediated transformation
Zea mays	ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4	Golden Gate	Protoplast and Agrobacterium-mediated transformation

Liang et al. recently demonstrated TALENs technology in maize. They designed five pairs of TALENs for targeting four endogenous loci, namely ZmPDS, ZmIPK1A, ZmIPK, and ZmMRP4, responsible for catalyzing phytic acid biosynthetic pathway. Each pair of TALEN-encoding construct was introduced into maize protoplast by PEG-mediated transformation and in maize embryo by Agrobacterium-mediated transformation. The transformation of these TALENs resulted in a group of INDELs, with targeting efficiencies of up to 23.1% in protoplast and about 13.3–39.1% in transformed plants. TALENs have also been used for targeting three homoalleles in hexaploid bread wheat. To modify all three (TaMLO-A1, TaMLO-B1, and TaMLO-D1) alleles, they designed a pair of TALEN targeting a conserved domain in the three alleles, to develop resistance against fungus-causing powdery mildew . They succeeded in creating fungus TALEN-free resistant plants by selfing for three generations one plant carrying a heterozygous mutation for each homoallele, underscoring the large opportunities for breeding programs offered by TALENs technology.

Another example highlighting the potential of such powerful technology includes the edition of tomato genome using TALENs to mutate PROCERA gene . For this purpose, they designed TALENs construct under the control of estrogen-inducible XVE promoter. Spraying of plants with estradiol gave rise to a very low level of mutation. Subsequently, they immersed the cotyledons of T1 plants in an estradiol-containing solution at weekly intervals. It was shown that the resultant transformed plants contained the desired mutations, which were stably inherited and that the TALEN construct was lost by segregation. TALENs have been successfully used to target several genes in different plant species such as Arabidopsis, tobacco, and Brachypodium for various applications . The details of the targeted gene(s) in different plant species via TALENs technology is given in Table .

9 Advantages of TALENS over other genome editing technologies

ZFNs, TALENs, and CRISPR/Cas9 are advanced genome editing tools that have wide significance for basic plant biology and can be used for multiplexed perturbation of gene networks of complex genomes . TALENs offer several potential advantages over ZFNs and CRISPR/Cas9 system. Even though ZFNs was the first programmable genome editing tool used, two major limitations prevent its wider applications. First, ZFNs are difficult to design and require intensive resources for new gene targets and second, their potentially dangerous off-target cleavage often leads to undesired mutations and chromosomal aberrations . TALE-based DNA-binding modules are found to be more efficient than zinc fingers because they are superior in specificity, confer high specificity, exhibit low off-target activity, and are less cytotoxic and in view of their expansive targeting range []. The major limitation of CRISPR/Cas9 system is that it targets the sequence preceded by 5′-NGG-3′ (known as protospacer adjacent motif) . TALENs target up to 16–24 repeats, while CRISPR/Cas9 system is restricted to 20 bp only. So, there ought to be higher possibilities of mismatches and off-target cleavage by Cas9. A high frequency of off-target mutations resulted by CRISPR/Cas9 system reported in human cells may seriously limit this technology . The other important advantage of TALENs over ZFNs and CRISPR/Cas is that it can efficiently target any small DNA sequences such as DNA sequences coding microRNA and enhancers, which may lack in targetable sites for ZFNs or CRISPR/Cas .

10 Concluding remarks

Over the last decade, new genome modification tools such as ZFNs, CRISPER/Cas, and TALENs have demonstrated the potential to perform microsurgery on genes, precisely modifying DNA sequence at exact location with ease. During the last few years, there has been tremendous advancement in TALEN technology in particular. Still, application of TALEN-mediated gene modification in plants is way behind compared to that in other organisms. There are many challenges that need to be addressed that would help TALENs engineering to be applied widely in plants. First, the bulky size of TALENs is likely to limit their broader application, particularly in cases where efficient gene modification cannot be achieved. Second, despite the findings of several novel RVDs that extended the scope of DNA recognition code thereby minimizing the off-target activity of TALENs, the efficiency of specific DNA targeting of TALENs needs still to be increased. Third, suitable methods for TALEN delivery into plant cells need to be evolved as TALENs-mediated gene engineering in plants requires efficient genetic transformation, in view of some plants being recalcitrant to transformation. Fourth, the mechanism of DNA repair should be studied in greater detail. Since, DNA repair by HR is usually limited by high rate of NHEJ, an in-depth knowledge of DNA repair process and the designing of new methods for enhancement of HR are required in order to achieve precise deletion, insertion, or replacement for introduction of new genomic traits in crop and other plant species. Finally, the notion that gene modification is limited only to basic research and cannot be realized in an industrial scale should be abandoned.

In conclusion, there still exists numerous problems regarding targeted genome modifications in plants as complete genome sequence information for many crops is still lacking and the accurate introduction of foreign DNA fragments into the host genome without perturbing them is a prime requirement for acceptance of GM crops. Though, TALENs molecular scissors technology is a major breakthrough technology in genome editing and has the potential to allay concerns about the random integration of foreign DNA, much work is needed to find ways to overcome the inherent problems before the technology is universally applied. Thus, TALENs technology opens the door to a new era of genetic improvement of plants overriding the negative aspects of existing plant biotechnology.

The authors have declared no conflict of interest.