There are two distinct strategies for the targeting of plant genomes (Figure 1). In the first, targeting is solely dependent on the use of NREs, while in the second, targeting also requires the delivery of a foreign donor DNA molecule. With the exception of single-monomer NREs (e.g. when targeting semi-palindromic sequences by ZFN or TALEN monomers or when using certain homing endonucleases), targeting experiments call for simultaneous expression of two or more NRE monomers in a single target cell. Delivery of two monomers can be achieved by co-delivery of two independent expression vectors, by using a dual-gene transformation vector or bicistronic transformation vectors. All of these vectors can potentially be used in transient and stable transformation experiments, with or without delivery of a donor DNA molecule. In addition, viral vectors have been developed for transient NRE expression in plant cells, as we describe further on.
Transient expression systems
In their pioneering study, Salomon and Puchta (1998) used Agrobacterium-mediated genetic transformation to transiently express I-SceI in transgenic tobacco plants and discovered that the I-SceI-encoding T-DNA molecules can integrate into the I-SceI-induced DSB. Whether it was the actual I-SceI-expressing T-DNA or another I-SceI-coding T-DNA molecule that co-entered the target cell and integrated into the break site was not determined, but in later reports (Chilton and Que, 2003; Tzfira et al., 2003), co-transformation of a donor DNA with homing endonuclease-expressing T-DNAs was used to target a second T-DNA (donor) molecule into the genomic DSBs (Table 2). The high frequency of non-selective NHEJ-mediated site-specific T-DNA integration (2.58%) (Tzfira et al., 2003) suggests that T-DNA molecules may be preferentially directed to genomic DSBs (Tzfira et al., 2004; Ziemienowicz et al., 2008; Dafny-Yelin et al., 2009). It is important to note that I-SceI- and I-CeuI-targeted plants (Chilton and Que, 2003; Tzfira et al., 2003) that were free of restriction enzyme-encoding T-DNA were recovered, indicating that using T-DNA molecules can be useful for transient NRE expression and delivery of donor DNA in plants.
Co-transformation of donor and ZFN-expressing DNA molecules was also used by Wright et al. (2006), who targeted and repaired a non-functional reporter gene by HR-mediated gene replacement in tobacco cells (Table 2). The authors first produced a collection of target transgenic tobacco plants, in which the non-functional gus::nptII, which was also engineered to carry a ZFN recognition site, was integrated at different genomic locations in different transgenic lines. Next, transgenic protoplasts were co-transformed with ZFN-expressing and donor plasmid DNA molecules and were selected for HR-mediated kanamycin resistance. The average frequency of kanamycin-resistant calluses was 4.5 × 10−3. The frequency of the kanamycin-resistant calluses was over threefold higher in co-transformation experiments of ZFN and donor DNA, compared with transformation of just donor DNA. Note that the authors recovered kanamycin-resistant callus in the absence of ZFN-expressing T-DNA as well, by promoter trapping of the nptII sequence. This experimental scheme, which allowed the authors to estimate the ratio between HR and NHEJ targeting events as 1:5.9, further validated the usefulness of co-transformation approaches for transient expression of NRE and delivery of donor DNA molecules into the target plants.
Co-transformation was also used in targeting experiments in BY2 tobacco cells, where a donor DNA was directed to a pre-integrated defective reporter gene as well as to the endogenous locus (Cai et al., 2009). For targeting of the transgenic locus, which was pre-engineered to carry disabled gfp and pat genes and was randomly integrated into the genome of BY2 tobacco cells, a single ZFN monomer-expressing T-DNA molecule was co-transformed with a donor T-DNA molecule into the transgenic BY2 lines. HR-mediated targeting events were selected and identified by replacement of the disabled gfp and reconstitution of a functional pat expression cassette. The latter derived from precise recombination between the donor and the disabled pat gene on the BY2 chromosome, as determined by isolation of bialaphos-resistant lines, Southern blot analysis and DNA sequencing of selected events. For targeting of a native locus in BY2 cells (i.e. the endochitinase gene CHN50), a pair of CHN50-specific ZFNs and a donor DNA were co-delivered into the target cells. To facilitate the transient co-expression of the two monomers of CHN50 ZFN in the target cells, the authors constructed a dual-gene expression cassette by flanking the 2A sequence with the CHN50 ZFN monomer-coding sequences and expressing the fusion under the control of the strong and constitutive CsVMV promoter. Here too, Agrobacterium-mediated genetic co-transformation was used for delivery of the CHN50-ZFN and donor DNA into BY2 cells, as well as into tobacco leaf disc cells. A dozen targeted bialaphos-resistant BY2 isolates and five transgenic tobacco plants were produced, and PCR and sequence analyses confirmed that they derived from HR-mediated gene replacement events. Although Southern blot analysis revealed that some of the targeted lines also carried randomly integrated donor DNA, these studies further supported the notion that co-transformation is a viable system for the delivery of NRE and donor DNA molecules into target plants. Indeed, the co-transformation approach was also used for co-delivery of dimer ZFNs, designed to target the ALS-encoding genes SuRA and SuRB in tobacco (Maeder et al., 2008, 2009). The enzymes were delivered as individual plant expression plasmids into tobacco protoplasts together with a third plasmid, carrying a constitutive kanamycin-resistant selection cassette, which was used to aid with the selection of transformed cells. Three out of 66 kanamycin-resistant transgenic plants carried a single-base deletion within the SuRA target sequence, but no SuRB mutants were detected (Maeder et al., 2008). This was later attributed not only to the design of the NRE but also to other possible factors such as chromatin structure and DNA methylation, as suggested in a study where both SuR genes were co-targeted using a different ZFN (Townsend et al., 2009).
Co-targeting of SuRA and SuRB in tobacco was not limited to detection of mutation events by pyrosequencing, but was also achieved by HR-mediated gene replacement (Townsend et al., 2009). Using the co-transformation approach, in which pairs of SuR-ZFN monomers and a third (donor) DNA molecule (which was partially homologous to the SuR loci, but carried specific mutations) were delivered into a target protoplast, HR-mediated targeted herbicide-resistant calluses and plants were recovered (Townsend et al., 2009). The estimated rate of ZFN-induced genome editing ranged between 2.4–5.3%, of which 0.2–4.0% was attributed to HR-dependent recombination between donor DNA molecules and SuR loci. Note that over 2% of the targeting events occurred more than 1300 bp away from the ZFN cleavage site (Townsend et al., 2009). Similarly, Cai et al. (2009) observed targeting events at a distance of 3000 bp from the ZFN-induced DSBs. Taken together, these observations suggest that a certain flexibility may exist when designing NRE for gene targeting. It is also worth noting that among the 47 different herbicide-resistant calluses obtained by co-transformation of donor and ZFN-expressing constructs, 19 were modified at multiple SuRA loci (by either HR or NHEJ) and 10 fully developed SuRA and SuRB double mutants were generated (Townsend et al., 2009). This study also demonstrated that co-targeting of similar genes can potentially be achieved by a single pair of ZFN monomers. Indeed, co-targeting of a duplicated gene using a single pair of ZFN monomers was later reported in soya bean (Curtin et al., 2011), as described further on.
HR-mediated gene targeting by co-transformation of a ZFN expression construct and donor DNA was also proven instrumental for targeting IPK in maize plants (Shukla et al., 2009). Maize plants carry two IPK paralogs (IPK1 and IPK2) that share 98% sequence identity in their coding sequences. Several ZFNs have been developed to target IPK1, selected for targeting based on its expression pattern. Deep-sequencing analysis of DNA extracted from maize cell culture transiently transformed by ZFN expression vectors was used for initial screening of the different ZFNs (Shukla et al., 2009). Two distinct IPK1-homologous donor DNA molecules were constructed: the autonomous donor molecule was designed to carry a complete pat expression system, while the non-autonomous donor carried a promoterless pat. Targeting by the non-autonomous vector was dependent on functional trapping of pat by the IPK1 promoter. Co-delivery of ZFN with donor DNA resulted in HR-mediated gene replacement and recovery of herbicide-resistant calluses in which one or both of the IPK1 alleles were targeted, as confirmed by molecular analysis (Shukla et al., 2009). Interestingly, the number of herbicide-resistant calluses was lower, while the frequency of HR-mediated targeting events (out of total targeted and random integration events) was higher for the non-autonomous donor than for the autonomous one. Furthermore, while calluses obtained from random integration events were characterized by multiple insertions, those obtained from HR-mediated integration events were characterized by a single, site-specific integration of the pat gene. As DSBs may act as ‘hot spots’ for NHEJ-mediated T-DNA integration (Tzfira et al., 2004; Ziemienowicz et al., 2008; Dafny-Yelin et al., 2009), we suggest that the ZFN expression construct may not have been delivered into the former cells, thus leaving the donor DNA molecules free to integrate at random, naturally occurring, but short-lived DSB sites. ZFN expression on the other hand may leave the DSBs available for integration for longer periods of time, and perhaps direct several T-DNAs to the break site, where only one T-DNA molecule will eventually integrate via HR. While we can only speculate on the possible mechanism by which donor DNA molecules were directed to HR and not NHEJ in targeted maize cells, these observations demonstrate the feasibility of obtaining HR-mediated targeted plants, which are free of additional, randomly integrated, foreign DNA molecules.
It should be noted that while IPK2 is nearly identical to IPK1, it remained intact in five independent IPK1-targeted plants, as determined by sequence analysis. In addition, sequence analysis of five more of the most probable off-target sites for the IPK1 ZFNs revealed that they were all true to type. These observations, as well as genotyping, segregation analysis and phenotypical characterization of mutant lines and their progeny, indicate that targeted plants with single and well-defined foreign DNA integration can be produced by transient expression of highly specific ZFNs and co-transformation of donor DNA.
Transgenic expression systems
A transgenic approach was used by Lloyd et al. (2005) who were the first to demonstrate ZFNs’ applicability for site-specific mutagenesis using a genomically inserted target site in Arabidopsis plants. Transgenic Arabidopsis plants were produced in which the QQR ZFN (Bibikova et al., 2001) was driven under the control of a heat-shock promoter. Heat treatment was used to induce QQR ZFN expression at specific developmental stages (i.e. in 10-day-old plants) and the presence of the EcoRI site within the ZFN recognition site facilitated the molecular analysis of targeting events, as already described. By comparing the number of DNA fragments with disrupted and true-to-type EcoRI sites (both amplified from undigested DNA extracted from heat-shock-induced plants), the authors estimated the rate of QQR-induced mutations to be as high as 0.2 per target. Furthermore, sequencing analysis suggested that most of the ZFN-induced mutations could potentially lead to functional gene knockout and that about 10% of the offspring from the heat-shock-induced plants carried mutations. T1-mutated seedlings were most likely obtained from mutations in the early-stage L2 cells of the shoot apical meristem of the heat-shock-induced plants.
Transgenic ZFN expression was also used by Tovkach et al. (2009, 2010), who developed a whole-plant DNA repair assay (Figure 3) and used it to demonstrate that in addition to small deletions and/or insertions, ZFN-mediated site-specific mutagenesis can also lead to single-nucleotide replacement at the break site (Tovkach et al., 2009). Single-nucleotide changes were also observed in tobacco and petunia plants, obtained by ZFN expression by viral vectors, as well as by transgenic expression of different ZFNs in Arabidopsis plants, as we describe further on (Marton et al., 2010; Osakabe et al., 2010; Zhang et al., 2010). Transgenic ZFN expression was also instrumental for induction of HR-mediated gene replacement in Arabidopsis when a donor DNA was delivered by flower-dip transformation into transgenic plants (de Pater et al., 2009). More specifically, a target locus, which was composed of functional pat and gfp genes as well as a unique recognition site for two ZFN monomers, was first incorporated into the genome of Arabidopsis plants (de Pater et al., 2009). The ZFN expression cassettes (driven by Rps5 tissue-specific, tamoxifen-inducible, or constitutive 35S promoters) were also stably integrated (either as single or dual ZFN expression constructs) into the target plants. Interestingly, only 2% of the cells of the transgenic plants that were engineered for ZFN overexpression (using the constitutive CaMV 35S promoter) were mutated (de Pater et al., 2009). This low rate of site-specific mutagenesis led the authors to examine whether retransformation of the ZFN-expressing transgenic plants with a donor DNA might lead to its integration via HR. Rps5-ZFN-transgenic plants were selected for HR-mediated targeting experiments, as the rate of site-specific mutagenesis in these plants (where ZFN expression was controlled under the Rps5 promoter, which is active in dividing cells and early embryos) was even lower than in 35S-ZFN-transgenic plants (de Pater et al., 2009). A donor DNA, which was engineered with partial homology to the transgenic target locus and also carried a hygromycin-resistant selection gene, was delivered into Rps5-ZFN by flower-dip transformation: 3000 hygromycin-resistant transgenic Arabidopsis plants were produced and screened by PCR for targeting events (de Pater et al., 2009). Two lines were targeted by HR, and a third line may have derived from a HR recombination event, followed by release and re-integration of the recombinant T-DNA into a different genomic location. The relatively high targeting frequency of 10−3 suggested that the strategy of delivering foreign donor DNA as T-DNA molecule into transgenic, ZFN-expressing plants might be useful for HR-mediated targeting. While the targeted lines also carried randomly integrated T-DNA molecules, these, as well as the ZFN-expressing cassettes, could potentially be segregated out by sexual crosses.
More recently, Zhang et al. (2010) and Osakabe et al. (2010) reported site-specific mutagenesis of native genes in Arabidopsis plants. Here too, the authors elected to use a transgenic approach, but used different types of promoters to express their ZFNs in the target plants. Zhang et al. (2010), who targeted ADH1 and TT4, produced hygromycin-resistant transgenic Arabidopsis plants in which the ADH1 and TT4 ZFNs were expressed under the control of an oestrogen-inducible promoter. For each target gene, a pair of ZFN monomers was expressed as an in-frame fusion with the T2A peptide. For induction of site-specific mutagenesis, T0 seeds were germinated in the presence of 17β-oestradiol; 10-day-old hygromycin-resistant seedlings were then tested for the presence of ZFN-induced ADH1 and TT4 mutations by PCR analysis and DNA sequencing. Deletions, insertions and a single-nucleotide substitution event were observed, with an estimated somatic mutation rate of 16% of the alleles for ADH1 and 7% of the alleles for TT4, as estimated by sequencing of cloned PCR fragments (Zhang et al., 2010). Mutated Arabidopsis plants were then produced by collecting seeds from 17β-oestradiol-induced T1 plants that were left to develop, grow and set seed: 11 of 16 (69%) of the T1 plants yielded adh1 mutants, of which 18% (2 of 11) were mutated at both alleles, as determined by segregation analysis of their progeny. In addition, 10 of 30 (33%) T1 plants yielded tt4 mutants, all of which were mutated in both alleles. The high rate of site-specific mutagenesis showed in this study could be attributed in part to the use of the strong 17β-oestradiol promoter, which can potentially reach higher expression levels than the 35S constitutive promoter (Zuo et al., 2000).
While a heat-shock-induced promoter yielded a lower frequency (10%) of mutated progeny (Zhang et al., 2010), it was still found useful for the production of abi4 mutant lines (Osakabe et al., 2010): Arabidopsis plants were genetically engineered to express the ABI4-ZFNs under a heat-shock-inducible promoter. The enzyme monomers were expressed as a single unit fused by the 2A peptide. Surveyor nuclease assay was used to detect mutations in somatic cells of nine ABI4-ZFN-transgenic lines, with an estimated rate of 0.26–2.86%. Interestingly, 70% of the cloned mutations were classified as substitution mutations, while the remaining 30% were classified as short (1–3 bp long) deletions. When allowed to grow, develop and set seed, two of the heat-shock-induced ABI4-ZFN-transgenic lines produced heterozygous mutant plants, as determined by Surveyor nuclease assay. More specifically, 7 and 3 of the 96 seeds surveyed for each line exhibited a single-base deletion or substitution mutation, respectively, from which T3 homozygous mutants were obtained.
More recently, Even-Faitelson et al. (2011) reported that ZFN expression under the control of EASE leads to site-specific mutagenesis, as determined by reconstruction of a mutated GUS gene. More specifically, the authors revealed that by confining ZFN expression to the egg cell (which is the target of the Agrobacterium T-DNA during flower-dip transformation, Ye et al., 1999), mutated Arabidopsis plants were recovered, which had been derived from targeting events occurring at early stages of embryo development. Clear advantages to this approach are that it does not necessitate exogenous stimuli for ZFN induction, and it restricts ZFN expression to the target tissue, thereby minimizing the risk of ZFN toxicity to the rest of the plant. Another important advantage pointed out by the authors is that as every mutant obtained by EASE-mediated ZFN derives from an independent germinal event, the use of this approach is likely to produce a wide range of new mutated alleles.
Stable NRE expression was also used for targeted mutagenesis in maize and soya bean, with a designed endonuclease and ZFN, respectively (Gao et al., 2010; Curtin et al., 2011). A single-chain homing endonuclease, composed of two designed I-CreI monomers fused into a single polypeptide, was expressed under the maize ubiquitin promoter in transgenic maize (Gao et al., 2010). A total of 781 transgenic plants were screened for putative mutations by PCR, of which 23 lines exhibited monoallelic, biallelic or a chimeric mix of mutations. Sequence analysis of selected T0 plants revealed a wide range of mutations, from deletions of just a couple of base pairs to large (up to 71 bp) deletions. Segregation and PCR analyses of progeny of selected T0 plants confirmed the authenticity and stability of the designed I-CreI-induced mutagenesis in maize. For targeting experiments in soya bean, ZFN-mediated mutagenesis was first assayed in transgenic Agrobacterium rhizogenes-induced hairy roots (Curtin et al., 2011). The roots were also engineered to express 2A-fused monomers of various ZFNs under the control of the oestrogen-inducible promoter. PCR analysis of digested DNA from 17β-oestradiol-induced roots and DNA sequencing of selected clones indicated that the transgenic root assay is efficient and reliable for analyses of ZFNs in soya bean tissues. Stable ZFN expression was also the method of choice for production of mutated soya bean plants: a binary plant transformation vector carrying ZFNs targeting two paralogs of DLC4 was used to produce transgenic soya bean plants in which ZFN expression was induced during in vitro cultivation. Two mutated plants were obtained, one carrying a mutation in DLC4a and the other in DLC4b. Both plants, which were either heterozygous or chimeric for the mutated alleles, were allowed to grow, develop and set seed; while the dlc4a mutant exhibited severe developmental abnormalities, the dlc4b mutant produced a large number of seeds and its progeny were successfully used for recovery of homozygous and heterozygous DLC4b mutant lines. Note that 1 out of 24 T1 heterozygous dlc4b lines was ZFN-free, as determined by PCR analysis, indicating that transgene removal can be achieved by segregation or sexual crosses.
A unique approach for ZFN-mediated transgene deletion was taken by Petolino et al. (2010) who crossed ZFN-overexpressing plants with target transgenic plants, which were engineered to carry a GUS expression cassette that was flanked by recognition sites for the ZFN. Both types of transgenic plants were homozygous for the transgene. A large number of T1 × T1 GUS-negative hybrid plants were obtained, with a frequency of about 35% for one particular cross. PCR and sequencing analyses confirmed that the GUS cassette had indeed been removed, as well as the presence of insertions and deletions at the DSB site. Segregation analysis of F2 progeny demonstrated the heritability of the gus gene deletion trait. This approach can potentially be applied for deletion of native target sequences and chromosomes, as well as in other sexually propagated plants. It is important to note that while deletion of artificial genes (Figure 1) can be achieved using a single ZFN enzyme or even a single ZFN monomer, deletion of chromosomal fragments and NHEJ-mediated native gene replacement may require the co-expression of four ZFN or TALEN monomers, which can be achieved by co-transformation of multiple T-DNA molecules into plant cells (Dafny-Yelin and Tzfira, 2007; Naqvi et al., 2010) or by assembly of polyprotein transformation vectors (Halpin et al., 1999; El Amrani et al., 2004). However, the low efficiency of co-transformation and the formation of complex integration patterns which are often associated with multi-T-DNA and multi-plasmid transformation systems (Windels et al., 2008; Ziemienowicz et al., 2008), and the complexity of assembling large polyprotein transformation vectors, may pose a technical challenge for co-transformation of more than two monomers into plant cells. More recently, Tovkach et al. (2009) described the assembly of a vector system for ZFN validation and expression, which enables the expression of up to four independent ZFN monomer expression cassettes from a single T-DNA molecule. The system, which was built on the basis of a modular plasmid assembly system (Chung et al., 2005; Tzfira et al., 2005; Tovkach et al., 2010), can be easily adapted for assembly of new ZFNs into plant expression vectors, for expression of ZFN monomers under the control of different promoters and for stable transformation using different types of plant selection markers (Figure 5). Furthermore, because ZFN and TALEN monomers share their basic structure, it is likely that this system can be modified and adapted for expression of multi-TALEN monomers, as well as for expression of single and dual peptide-modified homing endonucleases.
Figure 5. General structure of a modular multi-ZFN expression system. The system supports the assembly of ZFN expression cassettes (plasmids 2–5) with or without a plant selection expression cassette (plasmid 1) onto the T-DNA region of the plant binary vector pRCS2. The promoter and terminator regions on each expression cassette can be exchanged by any other regulatory sequences, as shown by the exchange of 35S promoter (35SP) and terminator (35ST) with Rubisco promoter (rbcP) and terminator (rbcT) or heat-shock promoter (hspP) and nopaline-synthase terminator (nosT). A PCR-amplified DNA-binding domain (ZFP) can be fused with the FokI domain (illustrated in Plasmid 2). Three selection cassettes (hpt, nptII and bar) can be used for the production of transgenic plants.
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