The DFR locus: A smart landing pad for targeted transgene insertion in tomato

Targeted insertion of transgenes in plants is still challenging and requires further technical innovation. In the present study, we chose the tomato DFR gene involved in anthocyanin biosynthesis as a landing pad for targeted transgene insertion using CRISPR-Cas9 in a two-step strategy. First, a 1013 bp was deleted in the endogenous DFR gene. Hypocotyls and callus of in vitro regenerated plantlets homozygous for the deletion were green instead of the usual anthocyanin produced purple colour. Next, standard Agrobacterium-mediated transformation was used to target transgene insertion at the DFR landing pad in the dfr deletion line. The single binary vector carried two sgRNAs, a donor template containing two homology arms of 400 bp, the previously deleted DFR sequence, and a NptII expression cassette. Regenerating plantlets were screened for a purple-colour phenotype indicating that DFR function had been restored. Targeted insertions were identified in 1.29% of the transformed explants. Thus, we established an efficient method for selecting HDR-mediated transgene insertion using the CRISPR-Cas9 system in tomato. The visual screen used here facilitates selection of these rare gene targeting events, does not necessitate the systematic PCR screening of all the regenerating material and can be potentially applied to other crops.


Introduction
CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) technology surpasses other genome editing tools such as zinc finger nucleases (ZFNs), meganucleases and TAL effector nucleases (TALENs) due to numerous advantages in terms of cost, ease of use and efficiency of targeting DNA sequences [1].
The CRISPR-Cas9 system is based on RNA-protein interactions involving a single noncoding guide RNA (sgRNA) and the Cas9 nuclease derived from Streptococcus pyogenes, aureus or thermophilus. Double-stranded breaks (DSBs) induced by the CRISPR-Cas9 system are repaired using either of two mechanisms: i) non-homologous end joining (either the classical non-homologous end-joining reaction C-NHEJ or an alternative end-joining reaction alt-EJ, also called microhomology-mediated end joining, MMEJ), that can be error-prone and introduce mutations including deletions or ii) homology-directed repair (HDR) that needs a repair template with homology to the targeted sequence and can lead to precise gene knock-in PLOS  activity has no negative impact on plant growth and fertility [36,38,39] makes the DFR gene a suitable target for CRISPR-Cas9 modifications and a potential landing pad for transgene insertion into the tomato genome.
In the present study, we report the precise deletion of 1013 bp in the DFR gene in WVA106, the genotype in which we plan to perform HR-mediated gene insertion. Homozygous lines carrying this mutation displayed the expected no-anthocyanin phenotype and were used for transgene targeted insertions at the DFR landing pad locus. In our strategy, the precise insertion of transgenes by HDR-DNA repair at the landing pad will lead to the recovery of a functional DFR gene and thus to restoration of anthocyanin biosynthesis that should be visible as a purple-coloured phenotype as soon as the plants are regenerating in vitro. Targeted insertion of a transgene in the tomato DFR landing pad was obtained for six independent explants after an Agrobacterium mediated-stable transformation with a single binary plasmid carrying both the CRISPR-Cas9 system and a DNA donor template. Our system, or proposed derivatives, provides a straightforward alternative for targeted insertion of transgenes in tomato and can be used to detect and improve HDR-mediated gene engineering.

CRISPR-Cas9 vector construction for deletion and HDR-mediated targeted insertion
The CRISPR-Cas9 vectors used in this study were previously described [41]. The CAS9 gene present in the pDe-Cas9 vector (kind gift from Holger Puchta, Karlsruhe Institute of Technology) is driven by the constitutive Ubi4-2 promoter from parsley [24]. The pDe-Cas9 plasmid was modified by exchanging the BASTA resistance cassette (1224 bp HindIII fragment) with a kanamycin resistance NptII cassette (1397 bp HindIII fragment amplified by PCR) from pK7WG2D [42] to produce pDe-Cas9-NptII or with a hygromycin resistance Hpt cassette (1787 bp HindIII fragment amplified by PCR) from pH2GW7 [42] to give pDe-Cas9-Hpt. The pDe-Cas9-Hpt vector was modified by introducing a second Gateway cassette, attR3-ccdB-attR4 in Eco53kI to generate the pDe-Cas9-Hpt-GT plasmid. Tomato genomic sequences for the U6 and U3 promoters were identified with the Basic Local Alignment Search Tool (respectively coordinates 92150950-92151262 chromosome 1 and 44489659-44489973 chromosome 6) using the Arabidopsis U6-26 snRNA (X52528) and U3B snRNA sequences (X52629) as queries [43,44].
Deletion in the DFR gene was performed using two sgRNAs. Guide RNAs targeting the tomato DFR gene (#Solyc02g085020) were chosen using the CRISPOR website (http://tefor. net/crispor/crispor.cgi) [45]. Two target loci were selected, one in exon 3 (sgRNA-DFR#1, expression driven by the U3 promoter) and one in exon 6 (sgRNA-DFR#2, expression driven by the U6 promoter) of the DFR gene. Constructs were designed to create a deletion of 1013 bp in the DFR gene. Two target loci were selected for gene insertion, one in the junction of the deletion obtained (sgRNA-DFR#3, expression driven by the U3 promoter) and one at the end of the exon 6 before the STOP codon (sgRNA-DFR#4, expression driven by the U6 promoter). The sequences of all the sgRNA are shown in S1 Table. The cassette which provided the donor DNA repair template was designed with left and right homologous arms, each corresponding to the 400 bp and 392 bp sequences flanking both sides of the 1013 bp DFR deletion, and the DFR previously deleted sequence associated with a NOS terminator (nopaline synthase) followed by the NptII gene under the control of the NOS promoter. The DNA donor template was flanked by the target sequences of the sgRNAs DFR#3 and DFR#4. The target sequence of DFR#4 was modified in the DNA donor template to avoid unwanted DSB. The sequence of the donor DNA repair template is shown in S1 Fig.

Agrobacterium tumefaciens-mediated transformation and regeneration experiments
The tomato (Solanum lycopersicum) WVA106 genotype was used for Agrobacterium tumefaciens-mediated transformation. The plants were grown in sterile conditions in a culture chamber with controlled temperatures of 22˚C/18˚C and a photoperiod of 16h/8h (day/night). Agrobacterium tumefaciens-mediated transformation of the WVA106 cultivar and T2-DFR64a lines (selected to be homozygous for the 1013 bp DFR deletion and without T-DNA insert) were performed as described in Mazier et al. (2011) [46] using cotyledon and leaf segments from 8-12 day-old seedlings and Agrobacterium tumefaciens strain C58 pGV2260 containing the binary vectors: pDe-Cas9-NptII-DFR#1-DFR#2 or pDe-Cas9-Hpt-GT-DFR#3-DFR#4-DFRtemp, respectively. Individual buds regenerating on 100 mg/L kanamycin-containing media were separated in culture tubes before molecular analysis. To obtain in vitro regenerated buds without transformation, cotyledon fragments were first placed on MS medium [47] containing 0.9 mg/L thiamine, 0.2 mg/L 2-4D, 0.1 mg/L kinetin in dark. After three days, they were transferred to regeneration media containing 2 mg/L zeatin until the buds had grown enough for phenotypic observation of DFR deleted plants.

DNA extraction, PCR genotyping and sequencing of regenerating plantlets
After the Agrobacterium tumefaciens-mediated transformation, leaf samples from kanamycinresistant regenerated plantlets were collected. Genomic DNA was extracted from fresh leaf samples according to Fulton and Tanksley [48]. The quality of the DNA was verified by Nanodrop and calibrated to a concentration of 500 ng/μl. To screen for the DFR gene deletion, T0 plantlets were genotyped using PCR with the primers DD1F and DD1R (S2 Table) designed to amplify a differentially sized fragment depending on whether the deletion is present or not (respectively 1347 bp and 2360 bp). PCR products corresponding to the deletion in the DFR gene were sequenced. Alignments were performed using Bioedit software [49].
The presence of the CAS9, NptII, and Hpt genes was verified by PCR using Cas9F and Cas9R primers (1438 bp fragment if positive) designed to amplify a region between the 3' end of the ubi4-2 promoter and the 3' end of the CAS9 gene, NptIIF and NptIIR primers (593 bp fragment if positive) and HptF and HptR primers (573 bp fragment if positive), respectively (primers are listed in S2 Table).
For detection of the HDR-mediated targeted insertion, T0 plantlets were screened by PCR with the primers GT1F and GT1R (1211 bp fragment if positive), designed to detect the 5' end of the targeted insertion, and with primers GT2F and GT2R (1338 bp fragment if positive) designed to detect the 3' end of the targeted insertion. All PCR products were resolved on 1.5% agarose gels at 120 V for 30 min.
In an attempt to amplify the whole reconstructed DFR gene associated with the insertion of the NptII gene by HDR, a long-range PCR strategy was performed with the PrimeStar GXL DNA polymerase from Clonetech. The primers, GT3F and GT3R, were designed to amplify differentially-sized fragments depending on whether the targeted insertion is present or not (3608 bp and 2180 bp, respectively) or if there is a deletion in the DFR gene (1167 bp) (S2 Table) Anthocyanin quantification by liquid chromatography For extraction, 2 grams of hypocotyls from 45 day-old seedlings and stems from 30 day-old in vitro regenerating plantlets were frozen in liquid nitrogen, ground and then lyophilized. Extraction solvent consisting of Ethanol/Water (70/30) was added to the dried samples and the extraction was performed by homogenization with an ultra-turrax and shaking at 4˚C. After centrifugation, the supernatant was collected and dried in a vacuum centrifuge. 1 ml of methanol was added to the dried sample and transferred to the chromatograph. The analysis was performed with a High-Performance Liquid Chromatography system, SHIMADZU Prominence, equipped with a reversed phase C18 column (MERCK Superspher RP18 endcapped) coupled with a photodiode array detector. The anthocyanins were characterized according to their spectra and compared to known standards. For quantification, a calibration curve was obtained by injection of known concentrations of cyanidin-3-glucoside.

T1 and T2 transgenic lines and greenhouse conditions
In vitro regenerated T0 plantlets were transferred to greenhouses after two weeks on rooting medium (MSO medium in which MS salts were reduced to ½). T1 and T2 transgenic lines were obtained by self-pollination of the T0 and T1 plantlets, respectively. Plants were grown in a sterilized peat soil mixture under natural light conditions with a daytime temperature of 22˚C and a night time temperature of 18˚C for the phenotypic observations.

Targeted deletion at the DFR locus
Although available for several tomato varieties, dfr mutated WVA 106 lines are not available in the tomato genetic resources (Vegetables Genetic Resources Center of UR 1052-INRA. https://www6.paca.inra.fr/gafl_eng/Vegetables-GRC/Our-Collections/Tomato-Collection/ The-Solanaceae-Genetic-Resources-Network). We designed two sgRNAs: sgRNA-DFR#1, targeting exon 3, and sgRNA-DFR#2, targeting exon 6, to obtain a deletion of 1013 bp at the 3' end of the DFR gene ( Fig 1A). The tomato WVA106 genotype was transformed via Agrobacterium tumefaciens carrying the pDe-Cas9-NptII-DFR#1-DFR#2 binary vector. Deletions in the DFR gene in kanamycin resistant regenerating plantlets were identified by PCR analysis ( Fig  1B). In parallel, PCR with primers specific for the CAS9 sequence was also performed on all kanamycin resistant regenerating plantlets. Out of 91 kanamycin resistant plantlets analysed, 52 carried the CAS9 gene and the DFR gene was deleted in 13 ( Fig 1B). Therefore, the DFR gene was successfully deleted in 25% of the plantlets carrying the CAS9 gene. The sizes of amplicons from some events were different from the wild type locus or the predicted deletion (T0 plants DFR47b, DFR13a and DFR31a for example). The amplification products corresponding to potential deletions in the DFR gene were sequenced and the results are shown in Fig 1C. Different deletion patterns were observed. The length of the deletions varied from 109 bp to 1355 bp. Target site 1 is missing in all deleted lines except for the DFR39a plant. Target site 2 was intact in nine plantlets out of the 13 analysed implying that the cutting efficiency was higher at the DFR targeted site 1. Two plantlets, DFR64a and DFR64b, contained the expected deletion of 1013 bp with the predicted Cas9 nuclease DNA cut three nucleotides upstream of the PAM motifs [50]. According to the PCR analysis several plants appeared to be homozygous for a deletion in the DFR gene with a unique PCR fragment (DFR55a, DFR31a, DFR87a, DFR83 and DFR91). In other plants, two PCR fragments (DFR64a, DFR64b, DFR39a, DFR68a, DFR88a, DFR47b and DFR13a) were amplified corresponding to the deleted and wild-type versions of the DFR locus. These plants are potentially heterozygote dfr mutants or chimeras consisting of mutant (heterozygotes or homozygotes) and wild-type cells. Plant DFR8a was clearly a chimera as three PCR fragments amplified for the DFR locus. Thus, 3.8% of the lines containing the CAS9 gene showed the predicted deletion. After transfer to the greenhouse, independent leaf samples were collected and DNA extraction and PCR analysis were repeated to confirm the presence of the deletion. The 13 plantlets showing a deletion in the DFR gene were self-pollinated. Two T0 plants, DFR55a and DFR31a, were sterile, thus T1 progeny of a total of 11 self-pollinated T0 lines were observed. The phenotype of the seedlings was recorded 10 days after germination. The T1 progeny from two T0 plants (DFR64a and DFR64b) showed a 1/4:3/4 segregation ratio for green versus purple seedlings (based on Chi2 test analysis, see S3 Table) characteristic of a deletion of the DFR gene present at the heterozygous stage in the T0 plants. For seven T0 progeny (DFR68a, DFR88a, DFR87a, DFR47b, DFR13a, DFR83 and DFR91) 100% of the plantlets were green. In comparison to the PCR analysis, this suggests that both alleles of the DFR gene were inactive, due to deletion at the homozygous stage (DFR87a, DFR83, DFR91) or bi-allelic modifications (DFR68a, DFR88a, DFR47b, DFR13a). Surprisingly, two T0 plants (DFR39a and DFR8a) produced 100% wild-type purple phenotype T1 plantlets. Molecular analysis on some of these plants failed to show any deletions in the DFR gene even though the deletion was detected in the T0 plants. In these cases, the deletions were probably not transmitted to the progeny due to the chimeric status of the parent.

Selection and phenotype of the homozygous dfr mutant line T2-DFR64a
PCR analysis of T1 progeny of the DFR64a T0 plant (containing the expected 1013 bp deletion), displaying the green phenotype characteristic of the homozygous dfr mutation, was used to identify plantlets where the T-DNA had segregated away. Selfing of these individuals produced T2 seeds that were homozygous for the deletion but without T-DNA and CAS9. One hundred percent of these T2 seedlings displayed the green phenotype associated with the dfr mutation. To evaluate the suitability of this "anthocyanin-free" phenotype for landing pad purposes and its potential usefulness at early stages of the transformation experiments, a regeneration experiment was carried out to compare these mutant plantlets with a wild-type control throughout in vitro culture (Fig 1D and 1E). Differences between the dfr mutants and the wild-type, based on the colour of the regenerating structures, particularly stems and leafstalks, were visible as early as one month after starting the regeneration experiment. In addition, the dfr mutation appeared to have no effect on the potential of the explants to regenerate, or on the growth or fertility of the plants.
Anthocyanin levels in the wild type and dfr mutant were quantified using high-performance liquid chromatography on hypocotyls from 45 day-old seedlings and the stems of 30 day-old regenerating events (Fig 1F). The anthocyanins were characterized according to their spectra and compared to known standards. Four compounds with spectra corresponding to anthocyanins were detected in the control extract at 540 nm. However, in the mutant extracts, these four compounds were not found. Total anthocyanins were quantified by addition of the areas of the four peaks. The value was calculated as an equivalent of cyanidin-3-glucoside according to the calibration curve. Anthocyanin content in μg equivalent cyanidin-3-glucoside per g of fresh weight was determined in the wild type hypocotyls and in vitro regenerating stems, with respectively 15.9 μg/g and 4.3 μg/g of fresh weight. Anthocyanin was not detected in the mutant extracts with a minimal level of detection of 0.05 μg/g.

Targeted gene insertion at the DFR landing pad
In order to easily integrate the DNA repair template in the same single binary vector carrying the CAS9 gene and the sgRNAs, we modified the pDe-Cas9 vector by inserting additional attR3 and attR4 Gateway sequences. This vector, named pDe-Cas9-Hpt-GT, allows for the simultaneous insertion of the DNA repair template (flanked by attL3 and attL4 Gateway sequences), and the sgRNAs (flanked by attL1 and attL2 Gateway sequences) during the LR reaction (S3 Fig). In order to induce targeted insertion at the DFR landing pad locus, associated with a restoration of the DFR gene, a donor DNA repair template was designed containing the missing DFR gene sequence (see M&M). To promote the insertion of the DNA repair template by homologous recombination, we included 400 bp and 392 bp long homology sequences identical to the 400 bp and 392 bp sequences flanking the deletion in the dfr mutant lines (Fig 2A). As proof of concept and to select regenerating buds in which the DNA repair template is integrated, we used the NptII gene as the inserted transgene. NptII was placed under the control of the NOS promoter and the 392 bp homology 3' arm sequence was placed immediately after its stop codon. Because this 3' homology sequence corresponds to the sequence present immediately after the DFR stop codon in the tomato genome, precise insertion of the NptII gene would potentially lead to it being under the control of the DFR terminator sequence. To restore DFR gene function, the endogenous DFR terminator was therefore replaced with a NOS terminator downstream of the DFR stop codon in the DNA donor template. In order to increase the frequency of gene targeting by the release of the donor DNA template in planta as previously proposed in Arabidopsis [24], the template was flanked by the target sequences of the sgRNAs DFR#3 and DFR#4.
The T2 DFR64a line was transformed with pDe-Cas9-Hpt-GT-DFR#3-DFR#4-DFRtemp, containing the DFR DNA repair template as previously described as well as two sgRNAs targeting the deletion junction sequence at the DFR locus of the DFR64a line. A total of 583 cotyledon pieces were treated and placed on regeneration media containing kanamycin for selection after the co-cultivation period. The first 597 regenerating plantlets issued from at least 265 independent regeneration events (issued from different cotyledons pieces) were systematically analysed by PCR to screen for targeted insertions. In parallel with this molecular screening, the colour of the regenerating material was noted during medium changes every two weeks. After three months (one month on selective media), a few dark-coloured regeneration structures could be observed on cotyledons (Fig 2B, 2C and 2D). A total of six independent purple coloured regenerating plantlets was obtained (events 161, 183, 303, 387, 463 and 524). Targeted transgene insertion at the DFR locus was confirmed by PCR in these six plantlets (Fig 2E and 2F and S4 Table). PCR analysis of the other 597 green coloured plantlets confirmed that the transgene had inserted randomly in these lines. The amplification products were sequenced and confirmed the targeted insertion at the DFR landing pad. Five out of the six events (T0 #161, 303, 387, 463 and 524) were issued from HDR on both sides ( Fig 2G) and PCR amplification of the full length DFR and NptII gene insertion block was obtained for events 463 and 524 ( Fig 2F). For event number 183, no amplification product was obtained with primers GT2F and GT2R (3' side) while PCR amplification of the 5' border was positive, corresponding to the reconstruction of the DFR gene. As 463 out of the 583 agro-infected cotyledons were able to regenerate kanamycin resistant plantlets, we can estimate that the efficiency of targeted insertion was 1.29% of the transformed explants.
The T1 progeny of four T0 plants with a targeted transgene insertion (# 183, 303, 387 and 463) were harvested, sown and the colour of their hypocotyls observed. For two events, T0 #303 and 183, hypocotyls of the T1 progeny (n = 50) were green, suggesting that the transgene was not transmitted from the T0 plant to the progeny. PCR analysis confirmed that the transgene was not present in these T1 plants. Non-transmission of the targeted transgene from the T0 plants to their T1 progenies could be due to the chimeric status of the transgene targeted insertion in the T0 plants #303 and #183. Hypocotyl colour and PCR analysis (not shown) in the T1 progenies (n = 81 and 66) of T0 plants #387 and 463 respectively, showed that the targeted transgene was transmitted as a Mendelian trait in these plants.

Discussion
The CRISPR-Cas9 system has been demonstrated to be an efficient and versatile tool in many organisms, including plants, for gene knock-out through NHEJ-mediated repair. In the present study, we confirmed previous reports on the efficiency of the CRISPR-Cas9 system to generate deletions in the tomato genome [3,21]. In our case, we obtained a 1013 bp deletion at the DFR locus with an efficiency of 25% by using a double sgRNA strategy. Two T0 plants showed exactly the expected and desired deletion in the DFR gene, and 11 other T0 transformants carried deletions ranging from 109 bp to 1355 bp. From these, 53.8% (7 out of 13) were bi-allelic or homozygous mutants.
We have shown here that deletion in the DFR gene at the homozygous stage led to a complete disruption of DFR function and to a seedling with green hypocotyls that could be easily visually discriminated from the purple coloured hypocotyl of the wild-type. These dfr homozygous line were the first step in our strategy aiming at using the DFR locus as a landing pad. We showed here that such mutant lines are easy to visually screen and obtain using a simple CRISPR-Cas9 strategy.
Next, using the dfr mutant line as starting material, we isolated six independent targeted insertions at the DFR landing pad. The rare HDR mediated events obtained after transformation were easy to identify due to the recovery of DFR function and the associated purple colour of the regenerated plantlets. The deleted DFR gene was repaired using a DNA template carrying the missing DFR sequences and 400 bp homology arms. Sequencing and molecular analysis of the purple-coloured T0 plantlets confirmed the precise HDR-mediated insertion of the DNA donor template and the recovery of the functional DFR gene.
In 1.29% of the transformed plants (6 out of 463) HDR-mediated insertion of the transgene occurred. This number is close to the frequency of HDR-mediated gene replacement obtained in Arabidopsis for example (frequency up to 0.8%) [51]. This low efficiency of targeted integration through Agrobacterium-mediated transformation was previously reported [24,27,41] and could be related to the low number of T-DNA copies delivered into the plant cells. Strategies based on particle bombardment or DNA virus-based replicons were reported to be valuable for HDR-mediated gene replacement in many plant species such as rice, maize, soybean, wheat, cotton and tomato, for reviews see [28,52]. Nevertheless, those direct DNA transfer techniques or genetically modified viruses are not necessarily accessible in all laboratories and delivery of DNA into the cell via Agrobacterium mediated transformation is still the method of choice for a great number of plants, including tomato. Different strategies have been proposed to increase the low frequency of HDR-mediated gene targeting through Agrobacterium transformation, such as inhibiting the NHEJ pathway by knocking-out the lig4 gene or using a positive-negative selection (PNS) system, for review see [31]. Unfortunately, the knock-out of genes involved in the NHEJ repair pathway can also have an impact on transformation stability and the capacity of the plant to repair DNA damage, leading to unwanted mutations. The PNS system involves the use of antibiotic resistance markers which are generally not desirable in crop breeding.
Other strategies based on the use of modified Cas9 (paired-nickase) or of the CPF1 nuclease have also been proposed to increase gene targeting frequency (8% to 14.5%) [24,41,53]. Finally, a strategy based on the release, via CRISPR-Cas9, of the donor template from the T-DNA in order to facilitate the accessibility of the donor template to the targeted locus has been described in Arabidopsis [24]. Here, we used this approach to increase the frequency of targeted insertions in tomato. We demonstrated that the combination of this strategy with the set-up of a visual screen, to easily detect the HDR-mediated gene insertion events, makes transgene targeted insertion feasible for crop breeding.
In our system, strict correlation was found between the restoration of a functional DFR gene, as revealed by a change in the colour of the plantlets, and the HDR-mediated gene targeting of the transgene at this locus. Thus, in addition to being an efficient landing pad for targeted transgene insertion, the DFR locus also provides an alternative endogenous reporter gene to detect HDR-mediated gene targeting and estimate its efficiency.
In conclusion, the strategy presented here has multiple advantages. The transgene targeting insertion is based on a simple and routine A. tumefaciens transformation experiment involving a single binary vector. Plants with a transgene targeted integration are easily selected by visual screening of regenerating seedlings. The DFR gene can be used as an HDR-based visual marker for gene knock-in improvements in which only a few bases must be changed. The DFR function is very well conserved in many plants, thus the proposed strategy is most likely applicable to other crop species.
Supporting information S1 Fig. DFR donor repair template used for HDR-mediated gene reconstruction. AttL3 and attL4 are colorized with purple, target sequence sgRNA dfr#3 is colorized with black and white letters, target sequence sgRNA dfr#4 is represented by grey color and black letters. The homology left and right arms are represented in yellow and orange. The deleted sequence is represented in dark yellow and white letters. The promoter Nos is represented in brown, and the terminator Nos is represented in pale brown and white letters. The NptII gene is represented in blue. The synonymous mutations designed to disrupt the sgRNA target sequence dfr are represented in red. (DOCX) S2 Fig. Backbones sgRNA-U3 and sgRNA-U6 used in tomato. In green, the tracrRNA motif, in orange and blue the promoter U6 and U3. In purple, the Gateway sequences attB1 and attB2. The stars represent the target sequence of the form 5'-A-N (19) NGG-3' with the respect to the U3 promoter and of the form 5'-G-N (19) NGG-3' with the respect to the U6 promoter. Underlined, restriction enzyme sites used for the cloning of the double sgRNA (XhoI -SalI). The PstI site is present in the pDONR207 sequence.