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. 2015 Oct 1;14(4):1151–1160. doi: 10.1111/pbi.12483

Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut

Katja Schneider 1, Andreas Schiermeyer 1, Anja Dolls 1, Natalie Koch 1, Denise Herwartz 1, Janina Kirchhoff 1, Rainer Fischer 1, Sean M Russell 2, Zehui Cao 2, David R Corbin 2, Lakshmi Sastry‐Dent 2, W Michael Ainley 2, Steven R Webb 2, Helga Schinkel 1, Stefan Schillberg 1,✉
PMCID: PMC11388843  PMID: 26426390

Summary

Genome modification by homology‐directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR‐mediated gene exchange of expression cassettes in tobacco BY‐2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7‐kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4‐kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR‐mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin‐resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN‐based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.

Keywords: direct DNA delivery, flow cytometry, gene targeting, homologous recombination, tobacco BY‐2 cell suspension cultures

Introduction

Plant biotechnology has become an integral part of global agriculture, as demonstrated by the steadily growing acreage used to cultivate transgenic plants, which reached 181.5 million hectares in 2014 (http://www.isaaa.org). Compared to traditional breeding techniques, the direct manipulation of the genome to insert, delete or modify genetic information allows the rapid and precise introduction of new traits. Genome modification has been frequently achieved using engineered nucleases as ‘molecular DNA scissors’ (Tzfira et al., 2012) for controlled genetic manipulation in model plant species and crops (reviewed by Gaj et al., 2013; Tzfira et al., 2012). The underlying principle is the promotion of homology‐directed repair (HDR) or nonhomologous end‐joining (NHEJ) at artificially introduced double‐strand breaks (DSBs).

The first targeted induction of DSBs was achieved using the natural meganuclease I‐SceI, which has an 18‐bp recognition site (Puchta et al., 1996). However, research has focused more recently on modular systems combining nonspecific nucleases with DNA sequence‐specific engineered guiding systems. Such systems include transcription activator‐like effector (TALE) DNA‐binding domains (Christian et al., 2010; Moscou and Bogdanove, 2009) attached to a restriction endonuclease, or RNA‐guided targeting of a Cas nuclease by prokaryotic clustered regularly interspaced short palindromic sequences (CRISPRs) (reviewed by Hsu et al., 2014). Recently, these techniques have also been applied in planta for various genome modifications (reviewed by Puchta and Fauser, 2014). Guiding systems based on multiple zinc finger motifs coupled to the nuclease domains (zinc finger nucleases, ZFNs) have already been used for several years and have been applied in many different plant species (Durai et al., 2005; Kim and Kim, 2014; Weinthal et al., 2010). The recognition of sequences flanking the target site by two ZFN monomers results in DSB formation by a functional nuclease dimer, as initially shown for FokI endonuclease coupled to three zinc fingers recognizing 9‐bp binding sites (Kim et al., 1996; Smith et al., 2000). Further ZFN engineering has resulted in more specific and efficient zinc finger motifs that include noncanonical linker sequences and up to six units in each module able to recognize binding targets up to 18 bp long, combined with an advanced matrix to predict the interactions among different subunits (Guan et al., 2002; Jamieson et al., 2003; Moore et al., 2001; Tan et al., 2003; Urnov et al., 2005). ZFNs have been successfully used to add genetic information to or delete it from the plant genome, for example to achieve trait stacking (Ainley et al., 2013; Cai et al., 2009), to delete previously integrated transgene DNA (Petolino et al., 2010) or to delete chromosome regions (Qi et al., 2013). Zinc finger binding motifs fused to a nuclease domain have been introduced into various species to evaluate ZFN activity or create artificial loci for genome modification. For example, the open reading frame of the major HIV coreceptor CCR5 has been disrupted using ZFNs, inhibiting CCR5 gene expression in human cells to confer resistance to HIV (Holt et al., 2010; Manjunath et al., 2013).

Gene exchange is a valuable strategy in medical as well as agricultural biotechnology, allowing the correction of mutations or the replacement of one gene with another gene of interest (Badia et al., 2014; Urnov et al., 2005). In plants, targeted genome alteration to date has mainly been restricted to deletions or insertions, for example disrupting the IPK1 locus in maize by inserting an expression cassette using a ZFN that cuts at a single site (single‐cut strategy) (Shukla et al., 2009). Gene replacement with a complete DNA cassette into an engineered locus has been achieved in soybean using the FLP‐FRT recombinase system (Li et al., 2009) and in tobacco with the R‐RS system (Nanto et al., 2005). Only one gene exchange approach mediated by nuclease‐introduced DSBs has been reported in Arabidopsis thaliana, demonstrating the excision of nptII using the I‐SceI endonuclease from a genomic location and simultaneous integration of a cassette to complement a truncated gusA gene present at the target site (Fauser et al., 2012).

Here, we report the targeted and coordinated excision of a heterologous expression cassette in tobacco BY‐2 cells using a single ZFN cutting at two sites (double‐cut strategy) followed by the HDR‐mediated replacement of the original 7‐kb insert with a 4‐kb donor cassette encoding multiple markers. Linear or circular donor DNA was co‐introduced into suspension cells by direct DNA delivery along with a plasmid encoding the ZFN, yielding 16 independent precise exchange events confirmed by Southern blot analysis and sequencing, 13 of which displayed fluorescent marker protein expression.

Results

Gene exchange strategy and generation of target lines

To obtain a flexible and rapid test system for the evaluation of targeted exchange events, we introduced a target cassette into wild‐type BY‐2 suspension cells (BY‐2 WT) resulting in target line C#86. The introduced target cassette contained two autonomous marker genes for selection and screening: acetohydroxyacid synthase (AHAS) and turbo green fluorescent protein (GFP). Two flanking homology sequences of 1504 bp (homology A) and 1739 bp (homology B) contained the CCR5 ZFN (Perez et al., 2008) landing sites (Figures 1a and 2a, top). Single‐copy integration of the target cassette was verified by quantitative PCR (qPCR, data not shown) and Southern blot analysis (Figure 1a,d). In addition, GFP protein levels were analysed by fluorescence microscopy and flow cytometry to confirm insertion at an active locus and the production of functional GFP (Figure 1b,c).

Figure 1.

Figure 1

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Establishment of target line C#86 (a) Target construct (see also Figure 2a) with Southern blot strategy: restriction sites for genomic DNA digest indicated, red bar represents the hybridization site for the probe. (b) C#86 phenotype under confocal laser microscope: bright light (left), green fluorescence (GFP targeted to the ER) (middle) and overlay (right). (c) GFP fluorescence of WT (black) and C#86 (green) protoplasts analysed by flow cytometry. (d) Southern blot confirming single insertion of the target cassette: C#86 genomic DNA digested with NdeI (left panel) or XbaI (right panel). CCR5: ZFN cleavage site.

Figure 2.

Figure 2

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ZFN‐mediated gene exchange by homology‐directed repair (HDR). (a) Exchange strategy: target cell lines contain the integrated target construct (top) which is replaced by the donor DNA construct (pDAB100375mod, middle) to obtain the predicted sequence after successful cassette exchange by HDR (bottom). Kanamycin resistance based on npt II expression indicates donor integration (targeted or random) whereas RFP expression and reconstitution of the DSM‐2 gene indicate targeted exchange. Homology regions A and B function as introns after donor integration. AtuMas, AtUbi3, CmAct2 and AtUbi10: promoters, AHAS : acetohydroxyacid synthase, npt II: neomycin phosphotransferase, RFP : red fluorescent protein, GFP‐KDEL : ER‐targeted turboGFP , DSM‐2: Dow Selectable marker 2 and solid arrows: primer annealing sites for nested ‘in‐out’ PCR at the expected insertion borders. Primer sites (open arrows) and the restriction enzymes to generate alternative linear donor constructs have been indicated. Not drawn to scale. (b) Screening for HDR events by ‘in‐out’ PCR: example shows second‐round PCR with two HDR events detected among 46 tested kanamycin‐resistant transformation events. The expected size of both amplicons is ~1500 bp. PC: positive control (plasmid), NC: negative control (water), asterisk: HDR candidate event, open triangle: PCR positive for RFP site only, filled triangle: PCR positive for DSM‐2 site only.

The target cassette also contained the 3' portion of the Streptomyces coelicolor Dow Selectable marker 2 (DSM‐2) gene downstream of the homology B region (Figure 2a). The encoded protein confers resistance to the phosphinothricin‐based herbicide, bialaphos (Lira et al., 2011). The corresponding donor cassette used to promote HDR contained a promoterless gene encoding red fluorescent protein (RFP), an autonomous gene encoding neomycin phosphotransferase (nptII) to confer kanamycin resistance and the 5' portion of the DSM‐2 gene. HDR therefore reconstitutes the full‐length DSM‐2 gene, with homology region B functioning as an intron, and brings the RFP gene in close proximity to the AtuMas promoter provided by the target sequence (Figure 2a, bottom). This strategy allows the isolation of targeted as well as random integration events and provides two markers for screening the HDR‐mediated cassette exchange, that is providing fluorescence and bialaphos resistance.

Transformation of target lines and the isolation of donor insertion events

Target cells were transformed with separate donor and ZFN constructs co‐introduced by particle bombardment. The pTRAkc::MTED vector (Kirchhoff et al., 2012), containing the nptII gene (analogous to the HDR donor construct), was used to determine transformation efficiency in reference experiments. Selection on kanamycin‐containing plates resulted in callus formation within 3 weeks post bombardment with 134 ± 195 events per bombarded filter (100 μL packed cell volume).

Targeted cassette exchange was facilitated by delivering the ZFN and donor cassettes at a 1:4 or 1:9 molar ratio in ten independent experiments. The ZFN construct was codelivered with the donor cassette in the context of a 10‐kb circular plasmid or as a 7‐kb linear donor fragment prepared either by enzymatic digestion of pDAB100375mod with NdeI and XhoI or by PCR amplification using 3'‐phosphorothioate primers to prevent exonucleolytic attack (plasmid, F NX and PCR in Tables S1 and S3). This resulted in similar transformation efficiency to the reference experiments described above, with 145 ± 26 kanamycin‐resistant calli regenerated per bombarded filter.

Identification of targeted HDR events by PCR

Transformation events growing on kanamycin‐containing medium were assumed to contain an integrated donor cassette resulting from either targeted or random insertion, or a combination of both. Therefore, we first screened for candidate events with HDR‐mediated cassette exchange by ‘in‐out’ PCR that spans the recombination junctions on extracted genomic DNA from kanamycin‐resistant callus tissue (Figure 2). The amplification of ~1500‐bp products using primers binding at the 5' and 3' borders of each recombination site indicated an exchange event (Figure 2a,b). From genomic DNA samples isolated from 1326 transformation events, 74 (5.6%) tested positive for both recombination junctions by ‘in‐out’ PCR (Figure 2b, asterisks). A smaller group of 26 callus clones (2%) tested positive only at the 3' border (homology B) (Figure 2b, filled triangle), whereas a larger group of 159 callus clones (12.2%) tested positive only at the 5' border (homology A) (Figure 2b, open triangle).

Continuous kanamycin selection resulted in the survival of 48 of the initial 74 HDR events (HREs) identified by ‘in‐out’ PCR, and these were investigated further by amplification of the complete reconstituted marker gene sequences. The presence of the complete RFP and DSM‐2 gene cassettes was confirmed by amplification products of approximately 3000 bp, covering the AtuMas promoter, homology region A and RFP coding sequence or the split DSM‐2 gene including the homology region B, respectively. Amplification products representing RFP and DSM‐2 were generated for 18 (38%) of the HDR candidates (data not shown), whereas seven further candidates generated only one of the expected products and 23 candidates did not produce either of the expected products.

Characterization of targeted gene exchange events

The 18 HREs with complete RFP and DSM‐2 expression cassettes confirmed by cassette‐specific PCR were analysed in more detail by sequencing the amplified products. Sequencing results confirmed the seamless HDR‐mediated integration of the donor cassette in all but two HDR events: in HRE42‐78, an additional guanine was inserted downstream of the RFP start codon causing a frameshift in the expression cassette (Figure S1), and in HRE64‐313, three target‐encoded codons replaced two donor‐encoded codons directly downstream of the start codon without altering the reading frame. Both variations could reflect incorrect DNA alignment during HDR process, as the target and donor sequence both contain a microhomologous sequence downstream of homology region A, representing the start of the RFP coding sequence in the donor (grey box in Figure S1).

In parallel to PCR and sequencing analysis, DSM‐2 expression was tested by cultivating the HRE cultures on bialaphos‐supplemented medium. We found that callus material from all 18 HDR events yielding a 3'‐border PCR product was also resistant to 2 or 10 mg/L bialaphos. The growth phenotype ranged from comparable to growth on kanamycin for the majority of calli to reduced size and brownish colour for a few HDR events, suggesting lower levels of DSM‐2. These data are consistent with the detection of the complete selection marker gene in the evaluated HDR events. On the other hand, up to 17% of candidate calli that tested negative in the ‘in‐out’ PCR could also grow on 2 mg/L bialaphos, representing false‐positive resistance. We observed a similar inconsistency with control constructs mimicking the reconstitution of DSM‐2 and enabling variable growth phenotypes (data not shown), indicating that selection conditions need to be adjusted to use this gene as an efficient selectable marker in BY‐2 cells.

Characterization of HREs by Southern blot analysis

AseI restriction fragments are diagnostic for the anticipated gene exchange event (Figure 3a) and were analysed in Southern blots with a probe recognizing the AtuMas promoter. As expected, no signal was detected in the lane representing BY‐2 WT genomic DNA with this strategy. A 3.3‐kb fragment representing the intact original target cassette was detected in target line C#86 and the control line C#86‐D, the latter generated by transforming C#86 with the donor cassette but not the ZFN plasmid, to obtain solely random insertions (Figure 3b, open arrowhead). Successful HDR‐mediated exchange should generate a 2.9‐kb fragment, which was indeed detected in all 18 HDR events, confirming successful cassette exchange in these lines (Figure 3b, black arrowhead).

Figure 3.

Figure 3

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Evaluation of targeted and random insertion events by Southern blot analysis. (a) Southern blot strategies to detect cassette exchange and random donor insertion events; red bars represent the hybridization sites for the probes used in (b) and (c), respectively. (b) Southern blot to confirm cassette exchange: lanes contain 8 μg genomic DNA extracted from liquid homology‐directed repair (HDR) event cultures after digestion with AseI. The radioactive probe hybridizes to the AtuMas promoter region at the 5' end of the target sequence. Detected bands at the indicated sizes represent the original target construct (3.3 kb, open arrowhead) or a precise HDR insertion (2.9 kb, black arrowhead). The bracket indicates a range of bands representing additional nonhomologous end‐joining events at the target site. (c) Southern blot to detect random donor integration: reprobed membrane from (b) with donor‐specific probe (arrowhead: donor integrated at target site).

In addition to the expected fragment representing the HDR event, AseI‐digested genomic DNA from most of the HRE lines contained a second fragment detected by the same AtuMas probe. We therefore reanalysed the single‐copy status of the target DNA in line C#86 using a panel of restriction enzymes combined with probes specific for the 5' and 3' regions of the transgene (Figure S2). This analysis revealed the presence of at least two duplication events at the 5' end of the target construct that had not been detected during the initial characterization of the target line.

The AseI‐based digestion and probing strategy we used (Figure 3) resulted in a fragment of the same size (3.3 kb) for the partial and the full‐length unmodified target cassette (e.g. HRE42‐133 and C#86), indicating the presence of the AseI site in AtUbi3 in at least one of the partial targets. The 3.3‐kb band in line C#86 is therefore comprised of two different fragments with the same size, and lines HRE42‐133, HRE46‐167 and HRE64‐166 contained the unmodified partial target in addition to the detected fragment representing the recombination event. Amplification by PCR of the target‐specific GFP coding region from genomic DNA of these HRE lines also confirmed the presence of the additional target sequences (results summarized in Table S1). The same PCR analysis also revealed the presence of a relocalized GFP gene upstream of the target cassette in HRE43‐7, despite the absence of a Southern blot signal. The digested genomic DNA from all other lines yielded a second band of different size (brackets in Figure 3), either smaller than 2.9 kb or larger than 5 kb (HRE49‐39). DNA fragments corresponding to these Southern blot signals were amplified from genomic DNA isolated from selected HREs 42‐78, 42‐174, 46‐113, 46‐115 and 46‐191, using PCR primers annealing outside of the insertion site (data not shown). The sequences of the products indicated that these DNA fragments represented imperfect exchange events: the 2.5‐kb product present in HREs 42‐78, 42‐174 and 46‐113 reflected the removal of the target cassette by ZFN activity followed by direct ligation of the resulting ends by NHEJ. In line HRE46‐115, part of the nptII coding sequence was inserted during NHEJ, yielding a larger fragment of 3.1 kb. In line HRE46‐191, part of homology region A was lost, resulting in a smaller fragment of 1.9 kb.

Distinct from all other lines, HRE64‐292 displayed only one band in the Southern blot indicating perfect gene replacement, yet sequencing of the RFP coding sequence revealed some point mutations coinciding with weak red fluorescence (Figure 4). The genomic DNA derived from HRE42‐129 was of poor quality (data not shown) so the presence of further bands in addition to those we detected cannot be excluded. In conclusion, recombination and donor integration were confirmed for all HREs by Southern blot analysis, yet the presence of a second partial target cassette in line C#86 led to detection of additional partial integration events.

Figure 4.

Figure 4

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Analysis of red fluorescent protein marker functionality in HRE lines. Flow cytometry profiles of WT, C#86 and HRE protoplast preparations showing the percentage of fluorescent protoplasts among 10 000 analysed in total. Red fluorescent cells indicate targeted insertion of the donor cassette; loss of green fluorescence indicates excision of the target cassette. Representative data from one experiment are shown. Asterisks represent homology‐directed repair events with fluorescence profiles indicating correct cassette exchange.

The analysis strategy described above does not detect random integration of the donor cassette elsewhere in the genome. Therefore, the Southern blot membranes were stripped and reprobed with a PCR product annealing to the RFP coding sequence present both in successful donor targeting and in random integrated donor events. The donor contains a single AseI restriction site, allowing the number of integration sites of the donor in each HRE to be estimated. Three HREs (HRE43‐7, HRE46‐191 and HRE64‐292) contained only the donor integrated into the target site (Figure 3c, arrowhead). Extra copies of the integrated donor construct were detected in approximately 80% of the HREs, ranging from only one to more than five integration sites (Figure 3c).

We also screened the HDR events to detect integration of the ZFN DNA in the genome of the HREs. PCR analysis amplifying part of the ZFN coding sequence revealed the presence of at least a portion of the construct in four HREs (Table S1).

Functionality assessment of RFP expression by flow cytometry

In parallel to molecular analysis, protoplast cultures were prepared from the HRE lines, which were analysed by flow cytometry to detect RFP expression (Figure 4, Table S1). Among the 18 HDR events tested, 13 showed RFP expression in >20% of the protoplasts including eight events (47%) with a fluorescence pattern representing precise cassette exchange, that is gain of red fluorescence and loss of green fluorescence (asterisks in Figure 4). Both green and red fluorescent protoplasts were absent in HRE42‐78 and HRE64‐313, where additional nucleotides were inserted during the exchange process (Figure S1 and Table S1). Furthermore, cultures of HRE42‐133, HRE46‐167 and HRE64‐166 showed concurrent green and red fluorescence, confirming the presence of the original target cassette as observed by Southern blot (Figures 3 and 4). Protoplasts from HRE43‐7 with the re‐integrated GFP gene showed strong green fluorescence but only weak red fluorescence.

In samples from HRE43‐7, the two HREs with no detected background modification (HRE42‐129 and HRE46‐191), as well as several other HDR events (e.g. HRE49‐112 and HRE64‐171), the percentage of red fluorescent protoplasts was clearly lower than the expected approximate 100%, despite the verification of correct insertion site sequences. The reduced RFP expression in these lines may be explained by cytosine methylation of the RFP gene cassette. The proportion of fluorescent protoplasts could be increased significantly by treating three candidate cultures with 5‐azacytidine, a cytidine analogue that prevents the methylation of newly synthesized DNA (Jones and Taylor, 1980) (Table S2). Although epigenetic modification is most likely the explanation for the loss of fluorescence, the co‐existence of cells lacking a targeted inserted donor cassette (but possessing a randomly inserted donor and therefore kanamycin resistance) in the same culture cannot be excluded. The fluorescence profile among HDR events was generally variable, again confirming the results observed in Southern blot experiments and implying the presence of epigenetic (e.g. silencing) as well as possible genetic (e.g. chimerism) heterogeneity.

To summarize, we analysed 1326 initial donor transformation events and identified 48 HRE candidates (3.6%) by ‘in‐out’ PCR, which could be maintained under kanamycin selection. We found that 18 of the candidate HREs contained the complete donor cassette at the target site with 16 (1.2%) showing seamless integration by molecular analysis and 13 expressing functional marker proteins (Figure 5).

Figure 5.

Figure 5

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Summary of homology‐directed repair (HDR) event characterization. Flow chart representing HDR event candidate isolation and characterization. Numbers in arrows indicate the references for percentage calculation.

Alternative direct DNA delivery method for gene exchange

We also wanted to demonstrate ZFN‐mediated gene exchange by electroporation as an alternative method of DNA delivery. The electroporation of target line protoplasts with the donor plasmid pDAB100375mod resulted in a transformation efficiency of 8% based on transient DsRed expression 40–72 h post‐transformation, about 20 times higher than observed after biolistic DNA delivery. These high transformation rates would allow population analysis, for example the determination of ZFN cutting rates by next‐generation sequencing. The efficiency of ZFN‐mediated gene targeting by HDR trended towards that achieved using particle bombardment. From 188 callus clones, ‘in‐out’ PCR revealed seven events positive for both borders, 10 positive solely for the left border and 22 positive solely for the right border. One of the seven events was confirmed to have undergone HDR by marker‐specific PCR and sequencing (data not shown). This event E1‐23 featured variations at the RFP start codon comparable with the incorrect insertion in HRE42‐78 and HRE64‐313 obtained by particle bombardment (Figure S1).

Discussion

Gene exchange can save considerable time and resources in comparison with the deletion of one gene and subsequent insertion of a second gene of interest. Gene exchange using designed nucleases requires the coordination of ZFN‐mediated cutting and homologous recombination at sites separated by a few to several kilobase pairs. In this report, verification of precise cassette exchange by Southern blot and sequencing across the insertion borders demonstrated seamless donor insertion in 1.2% of the events isolated from a pool of kanamycin‐resistant transformants. Despite the large size of the exchanged DNA fragments, this is comparable to the efficiency of HDR events generated by a single ZFN cut (Ainley et al., 2013; D'Halluin et al., 2013; Townsend et al., 2009; Wright et al., 2005) or exchange of two marker genes by ZFN‐mediated NHEJ (Weinthal et al., 2013). At this efficiency, several exchange events can be obtained in medium‐scale experiments. Although data were more limited, we obtained similar results with a protoplast‐based transformation approach, expanding the possible utility of gene exchange approaches using ZFNs.

In other organisms and to a limited extent in plants, recombinase‐mediated cassette exchange (RMCE) has commonly been used to exchange DNAs for genome engineering with variable efficiency (e.g. Li et al., 2009; Louwerse et al., 2007; reviewed by Turan et al., 2013). An advantage of ZFNs and some other types of designed nucleases is that a large number of nucleases can be designed and utilized to target any endogenous or engineered locus (Ainley et al., 2013). It also allows flanking numerous genes in a stack with unique sites for designed nucleases to allow later genomic editing.

Engineering of transgenic loci in plants, in particular gene stacking, requires the capability to insert DNAs large enough to contain entire gene expression cassettes. Examples of such large targeted DNA insertions are limited in plants. Insertion of protein coding sequences alone has been reported using HDR (Shukla et al., 2009; 1.0 kb) or NHEJ strategies (Weinthal et al., 2013; size not specified). Insertion of entire gene cassettes has been demonstrated by ZFN‐mediated HDR for donor constructs of 2.7 kb and 2.4 kb (Ainley et al., 2013; Shukla et al., 2009), by meganuclease‐mediated HDR for a 6.6‐kb cassette (D'Halluin et al., 2013), for a 12.8‐kb donor cassette using the FLP‐FRT recombinase system (Li et al., 2009) and for a ~5‐kb donor cassette using the R‐RS recombinase system (Nanto et al., 2005). An exchange of a target sequence (nptII cassette, ~1.5 kb) with a donor construct (~1 kb) to complement a truncated gusA fragment present in the engineered target was reported by Fauser et al. (2012). The DSBs were induced at both sites of the exchanged fragments using the I‐SceI meganuclease in A. thaliana plants. With the exception of the Weinthal et al. (2013) report, which used Agrobacterium‐mediated delivery, particle bombardment was used to transfer the DNA into the plant cells. The largest DNA comprising functional genes targeted by HDR in higher eukaryotes is a 21‐kb DNA in a mammalian system (Jiang et al., 2013). Our studies describe an exchange by HDR of a 7‐kb DNA with a 4‐kb DNA, both of which carry full gene cassettes. Given the high rate of gene exchange we observed and the reports of successful targeting with larger DNAs listed above, the exchange of larger DNAs seems readily feasible technically and operationally using HDR and appropriate plant materials for transformation.

Kanamycin resistance as a first level of selection allowed us to isolate candidate events regardless of the nature of target cassette integration (targeted or random). We found that 80% of the HDR events were accompanied by one or more copies of randomly integrated donor DNA and in 22% of the HDR events, ZFN DNA had also integrated randomly into the genome. Biolistic delivery often results in multiple insertions in untargeted transformation experiments. In contrast to our data, a larger fraction of 30–40% of targeted insertion events in maize cell suspension cultures contain no trace of randomly integrated donor DNA, with similar values for particle bombardment and Agrobacterium‐mediated transformation (D'Halluin et al., 2008). While random donor DNA insertions make initial analysis of targeted events more challenging, with transformation systems in which fertile plants can be regenerated, random DNA copies are expected to occur less frequently. In addition, with the exception of events integrated in proximity of the target site (Chawla et al., 2006), unwanted random integration events can generally be readily segregated by breeding, when producing desired transgenic events for research or commercial transgenic events.

Heterogeneity in the event background and RFP expression led us to re‐investigate the target line C#86. In contrast to the results originally obtained during target line establishment, we detected additional partial copies beside the single insertion of the complete target cassette. Cell line C#86 was established ~4 years prior to the experiments described in this article. The partial duplication may have occurred during prolonged cultivation (reviewed in Phillips et al., 1994) given that karyotype variation is often detected in individual cells of one BY‐2 cell suspension culture (Kovarik et al., 2012). The presence of partial target loci copies might have led to a higher number of candidates tested positive only in ‘in‐out’ PCR at homology A, but would not compromise the validity of targeting frequency obtained in our experiments as all targeted events contain the fragment size expected in Southern blot analysis for targeted donor insertion.

In accordance with experiments performed in sugarcane embryogenic callus (Jackson et al., 2013), we observed no statistical difference in transformation efficiency or event quality when we compared plasmid and linear donor constructs (Table S3). Data obtained in mammalian cells suggest an influence of the donor format, as the usage of supercoiled circular donors resulted in approx. 60% higher targeted integration (Orlando et al., 2010). Advantages of linear donor DNAs include the elimination of bacterial vector integration at random sites and higher amounts of donor DNAs that can be used for targeting experiments.

The data obtained suggest that the C#86 target locus or specific construct elements (e.g. methylated promoter elements) affect the expression of the RFP (as demonstrated by the increase of expression in the azacytidine‐treated samples). Epigenetic changes are also a possible explanation for the unexpected high number of events that tested positive by ‘in‐out’ PCR but that could not be maintained under kanamycin selection, as 26 of the 74 initial candidates (35%) did not survive selection. Random full or partial copies of the insert could account for the variation of gene expression at the targeted locus, either by increasing gene silencing or by increasing the level of RFP expression if the promoterless RFP gene in the donor inserts behind an endogenous promoter. Silencing of targeted loci in plants might follow different mechanisms as in cultured cells; therefore, the design of a targeting strategy should aim at minimizing induction of silencing, for example by avoiding multiple copies of gene expression elements in the construct and promoter elements known to be susceptible to methylation (Day et al., 2000).

In addition, part of the expression patterns observed which are not expected by a precise DNA exchange alone could be explained by chimerism of the transgenic calli. The similar strength of the observed bands in Southern blot analysis (Figure 3b) and the presence of exactly two bands in all lines, however, indicate that the duplication of the target site persists in a single cell. In the case of chimerism, cell lines can be obtained from some heterogeneous cell suspension cultures by flow sorting based on fluorescent marker gene expression (Kirchhoff et al., 2012).

Our results confirm the excision of a target cassette by the CCR5 ZFN cutting at the CCR5 ZFN binding sites, as well as the HDR‐mediated seamless replacement of the target with a donor cassette, in tobacco BY‐2 cells. The target and donor cassettes allow multigene exchange in a commercially relevant context. Our data obtained in tobacco BY‐2 cells therefore suggest that ZFN technology is suitable for engineering transgenic loci by the simultaneous removal of undesired or less effective genes and the addition of other genes of interest in a wide range of crop species.

Experimental procedures

Plasmids

The target vector pDAB100355 contains a modified version of the manopine synthase promoter (Barker et al., 1983) from A. tumefaciens, homology region A derived from an intron sequence of the AMP activated kinase coding gene from A. thaliana (At5G21170), the acetohydroxyacid synthase (AHAS) from Gossypium hirsutum (Rajasekaran et al., 1996) under the control of the ubiquitin 3 promoter from A. thaliana (Norris et al., 1993), the TurboGFP gene from Pontellina plumata (Evrogen, Moscow, Russia) under the control of the melon (Cucurbita melo) actin 2 promoter (Clendennen et al., 2003), homology region B derived from an intron sequence of the coumaroyl CoA synthase coding gene (At3g21320) from A. thaliana and the 3' end of DSM‐2 (Lira et al., 2011) encoding a gene that confers resistance to bialaphos herbicide, derived from S. coelicolor (Figure 2a).

The donor vector pDAB100375mod (Figure 2a) contains homology region A, the TurboRFP gene from Entacmaea quadricolor (Evrogen), the neomycin phosphotransferase (nptII) gene (Bevan et al., 1983) under the control of the nopaline synthase promoter (Pnos) (Depicker et al., 1982) and the 5' part of the DSM‐2 selection marker under the control of the ubiquitin 10 promoter of A. thaliana (Norris et al., 1993) followed by homology region B.

DsRed encoded in reference plasmid pTRAkc::MTED is under control of a double‐enhanced 35S promoter and targeted to the plastids using the GBSS transit peptide (Kirchhoff et al., 2012). Plasmid pDAB100377 mimics the exchange at the 5' border containing the RFP gene downstream of the target AtuMas promoter and the homology A intron of AMP activated kinase. The selection marker encoded in pDAB100377 is AHAS.

Linear versions of the donor construct were produced by either enzymatic digest with restriction enzymes NdeI and XhoI or phosphorothioate‐protected primer pair COU‐intron_for‐PTO and AMP_intron_rev‐PTO (Table S4).

The ZFN monomers were expressed from pUC19‐9607 as a single open reading frame connected with a self‐cleaving 2A peptide sequence under the control of the Cassava vein mosaic virus (CsVMV) promoter.

Plant material and culture conditions

Tobacco (Nicotiana tabacum L) BY‐2 cells were cultivated in Murashige‐Skoog (MS) basal medium as previously described (Schinkel et al., 2008). The cells were subcultured twice weekly by transferring 5–7.5 mL of the culture into 50 mL fresh medium. Working cultures were inoculated with 2–10% (v/v) of 3‐day‐old cultures and were used 3–4 days after inoculation for particle bombardment and up to 7 days after inoculation for electroporation.

Callus material from HDR events was used to establish liquid cultures in 5–8 mL MS medium containing 1 mg/L kanamycin in 50‐mL TubeSpin® Bioreactors (TPP Techno Plastic Products AG, Trasadingen, Switzerland). After initial subculturing in the bioreactor tubes, cultures were transferred to 50‐mL Erlenmeyer flasks and subcultured once weekly using an inoculum of 3–10% (v/v).

Agrobacterium‐mediated generation of target lines

Target vector pDAB100355 has been introduced into A. tumefaciens strain LBA4404 (Invitrogen, Karlsruhe, Germany) by electroporation (Dower et al., 1988). Transgenic BY‐2 cells were generated by cocultivation as described (An, 1985). Transgenic events were selected on MS agar plates supplemented with 1.5 μM imazethapyr (Sigma Aldrich, Deisenhofen, Germany).

Particle bombardment of BY‐2 cells

Biolistic DNA delivery was carried out using the Biolistic PDS‐1000/He™ Particle Delivery System (Bio‐Rad Laboratories, München, Germany). BY‐2 cell aliquots of 100 μL packed cell volume (PCV) from working cultures were collected on 5.5‐cm filter discs (MN 615, Macherey‐Nagel, Düren, Germany) and incubated on solid MS medium or MS medium supplemented with 0.032 m mannitol and 0.032 m sorbitol at room temperature. Donor and ZFN plasmid DNA were used at a molar ratio of 4:1. The HDR constructs and control plasmid pTRAkc::MTED (5 μg) were coated onto 0.6‐μm gold microcarriers (1.5 mg gold/50 μL H2O) by precipitation with an equal amount of 2.5 m CaCl2 and 10 μL 0.1 m spermidine and were then washed and dispersed in 120 μL 100% (v/v) ethanol for 10 shots. Bombardment was performed with 10 μL aliquots per macrocarrier using a helium pressure of 650 psi and flight distance of 9 cm. After bombardment, cells recovered on the filters for 2–3 days at room temperature in the dark. Cells from each filter were collected into six‐well plates containing 2 mL MS medium without antibiotics. For transformation with the control plasmid, 250 μL aliquots were analysed, that is DsRed‐positive cells were counted using an automated upright microscope (Leica DM6000 B, Leica Microsystems, Wetzlar, Germany) to calculate initial transformants per filter. The remaining cells were plated. For all other transformations four aliquots of 500 μL were plated on selective MS medium (100 mg/L kanamycin). Plates were incubated for up to 4 weeks at 28 °C in the dark for callus formation.

Electroporation of BY‐2 protoplasts

Protoplasts were prepared as previously described (Schinkel et al., 2008) with modifications. BY‐2 cells were centrifuged (5 min, 1500 g ) and incubated in PNT‐medium containing 2% (v/v) Rohament cellulase, 1.5% (v/v) Rohament pectinase, 0.3% Rohapect UF (all AB Enzymes, Darmstadt, Germany) and 0.3% macerozyme R‐10 (Serva, Heidelberg, Germany) at 26 °C overnight. Protoplasts were isolated as described (Kirchhoff et al., 2012) except that the protoplasts were resuspended in electroporation buffer (Miao and Jiang, 2007) with 0.4 m mannitol instead of sucrose. Protoplasts were adjusted to 1 × 106/mL before transformation, and 600 μL aliquots were mixed with 60 μg plasmid DNA in cold 0.4‐mm electroporation cuvettes. Electroporation mixtures were set up containing donor DNA, ZFN‐DNA, donor DNA plus ZFN‐DNA (at a 9:1 molar ratio) or pTRAkc::MTED. The cuvettes were kept on ice until electroporation with a Gene Pulser Xcell™ Electroporation System (Bio‐Rad Laboratories) using one exponential pulse of 200 V for 70 ms. Transformed protoplasts were incubated in the cuvettes at room temperature for 30 min, then distributed into 3 mL 8p2c medium (Kirchhoff et al., 2012) and incubated at 26 °C.

After 40–70 h, the cells were analysed using a Leica DM6000 B microscope for survival and transformation efficiency. The cells were incubated in fresh 8p2c medium for 14–16 days at 26 °C before they were plated on selective medium (100 mg/L kanamycin).

Selection of transformation events

Initial resistant transformation events were isolated on 100 mg/L kanamycin 2.5–4 weeks after particle bombardment or 4–5 weeks after electroporation and were maintained on the selective medium in a 3–4 week subculturing routine. Genomic DNA was extracted from callus material for PCR and Southern blot analysis, and the candidates were also cultivated in 24‐well plates containing 2 mg/L or 10 mg/L bialaphos to identify the events positive for DSM‐2 expression.

PCR analysis

Genomic DNA was extracted from 100 mg of callus material using the NucleoSpin® 96 Plant II kit (Macherey‐Nagel) or M‐PVA Magnetic Beads (PerkinElmer chemagen, Baesweiler, Germany). The integration of the donor cassette was evaluated using a nested PCR approach with first‐round primer sets Target_RB_For3 and Donor_RB_Rev for the 5' border and Donor_LB_For3 and Target_LB_Rev3 for the 3' border and second‐round primer sets Target_RB_For2 and Donor_RB_Rev for 5' border and Donor_LB_For and Target_LB_Rev for the 3' border. PCR was carried out using the i‐MAX II polymerase kit with an annealing step of 30 s at 61 °C for 40 cycles in the first round and 25 cycles with the same conditions in the second round using 2 μL of the first PCR as template.

Extended PCR analysis to amplify the RFP and DSM‐2 cassettes was performed on genomic DNA extracted from 1 to 2 g fresh weight of suspension culture cells. PCR was carried out as above using primer pairs HS_Target_for and HS_Donor_rev for the RFP fragment and KS_LB_amp_fw1 and KS_LB_amp_rev2 for the DSM‐2 fragment. The PCR comprised an annealing step of 10 s at 55 °C for 40 cycles. The PCR products (approx. 3000 bp for RFP and DSM‐2, respectively) were sequenced for verification of the HDR events.

Southern blot analysis

Ten micrograms of genomic DNA was digested with AseI, and the DNA fragments were separated by electrophoresis in a 0.6% (w/v) agarose gel in TAE buffer for 3 h at 60 V. The gel was sequentially subjected to depurination (0.25 m HCl for 15 min), denaturation (0.5 m NaOH and 0.5 m NaOH/1.5 m NaCl for 30 min each) and neutralization (1 m Tris, 1.5 m NaCl, pH 7.0 for 30 min). The DNA was transferred to a positively charged nylon membrane (Roth, Karlsruhe, Germany) by vacuum blotting using 2x SSC buffer and applying 60–100 mbar vacuum pressure for 2–3 h (Vacu‐blot system, Biometra, Göttingen, Germany). Transferred DNA was linked to the membrane by baking (2 h, 80°C). Hybridization was performed with a 633‐bp probe binding to the AtuMas promoter or a 1030‐bp probe binding to the RFP coding sequence (Figure 4). The radioactivity on the Southern blot was analysed by exposure to a phosphoimager plate that was analysed with imager CR 35 Bio and the AIDA image analysis suite (Raytest, Straubenhardt, Germany).

Flow cytometry analysis

Protoplasts were prepared as described for electroporation but resuspended in 8p2c medium. Red and green fluorescence were analysed using a BD influx cell sorter (BD Biosciences, Heidelberg, Germany) equipped with a 200‐μm nozzle. The cytometer was operated using a 488‐nm laser (200 W) for GFP excitation or a 561‐nm laser (150 W) for RFP excitation and filter sets at 530/40 and 585/29 nm, respectively, for the corresponding emissions. WT BY‐2 cells were transformed with pDAB100377 to generate a reference RFP‐positive line (RFP+). Three types of protoplasts were used to set the gates for the presence of red and green fluorescence and to compensate for spectral crossover: WT BY‐2 protoplasts, protoplasts from line C#86 and RFP+. We analysed 10 000 gated protoplasts for each HDR event and processed the signal data with FACS sort software (BD Biosciences).

Supporting information

Figure S1 Sequence analysis of imprecise HDR.

Figure S2 Extended analysis of target insertions in C#86 target line.

Table S1 HDR event characterization.

Table S2 Flow cytometry analysis of HDR events after azacytidine treatment.

Table S3 Transformation and HDR efficiencies for single experiments.

Table S4 Primer sequences.

PBI-14-1151-s001.docx (5.2MB, docx)

Acknowledgements

We are grateful to Dr. Flora Schuster and Simon Vogel for technical assistance in cell culture maintenance and cytometer use. The authors thank Dr. Richard M. Twyman for his support with editing of the manuscript. We also thank colleagues at Dow AgroSciences for providing insightful comments for the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Sequence analysis of imprecise HDR.

Figure S2 Extended analysis of target insertions in C#86 target line.

Table S1 HDR event characterization.

Table S2 Flow cytometry analysis of HDR events after azacytidine treatment.

Table S3 Transformation and HDR efficiencies for single experiments.

Table S4 Primer sequences.

PBI-14-1151-s001.docx (5.2MB, docx)

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