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. 2015 Nov 6;16:232.
doi: 10.1186/s13059-015-0796-9.

High-frequency, precise modification of the tomato genome

Tomáš Čermák  1 , Nicholas J Baltes  2 , Radim Čegan  3 , Yong Zhang  4 , Daniel F Voytas  5
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High-frequency, precise modification of the tomato genome

Tomáš Čermák et al. Genome Biol. .
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Abstract

Background: The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.

Results: Here, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences.

Conclusions: High-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.

Figures

Fig. 1
Gene targeting with geminivirus replicons. a Structure of the bean yellow dwarf virus (BeYDV) genome. The single-stranded DNA genome encodes three major functions: replicase proteins (Rep and RepA) mediate rolling circle replication, and movement and coat proteins are essential for viral movement. The long intergenic region (LIR) is the origin of replication and also functions as a bidirectional promoter that drives expression of viral genes. The short intergenic region (SIR) is the origin of C-strand synthesis and contains transcription termination and polyadenylation signals. b Structure of BeYDV genome modified for gene targeting. Coding sequences for movement and coat proteins were replaced with the site-specific nuclease and donor template for gene targeting. The modified virus is not capable of infection due to the lack of essential viral proteins. Further, the size exceeds the limit for successful packaging and cell-to-cell movement. The replication function is preserved, and the vector can replicate when delivered to plant cells by transformation. c Illustration of gene targeting with the modified BeYDV vector through Agrobacterium-mediated transformation. The BeYDV genome, containing the nuclease and donor template for gene targeting, is cloned into a transfer DNA (T-DNA) vector. One LIR is placed on each side of the viral genome to ensure release from the T-DNA in the plant cell. During Agrobacterium infection, linear T-DNA molecules are delivered to the nucleus of a plant cell, where the viral genome is replicationally released in a circular form and amplified into thousands of copies by rolling circle replication, mediated by the replicase proteins expressed from the LIR. The nuclease expressed from the viral genome induces DSBs at the target locus, and the donor template is copied into the target site by homology-directed repair. The high copy number of donor templates increases the frequency of gene targeting. LB left T-DNA border, SSN sequence-specific nuclease, RB right T-DNA border
Fig. 2
Gene targeting upstream of the ANT1 gene. a Top: illustration of the GT event. Upon cleavage by the nuclease and homologous recombination with the replicon, the donor cassette is inserted upstream of ANT1. Bottom: structure of the transfer DNA (T-DNA) vector, pTC144, which produces DNA replicons. LB left T-DNA border, LIR BeYDV large intergenic region, 35S cauliflower mosaic virus 35S promoter, tHSP Arabidopsis thaliana heat shock protein 18.2 terminator, SIR BeYDV short intergenic region, REP coding sequence for Rep/RepA, RB right T-DNA border. Additional components of the donor include: NosP Agrobacterium tumefaciens nopaline synthase promoter, NPTII neomycin phosphotransferase gene for kanamycin resistance, t35S CaMV 35S terminator. For expression of CRISPR/Cas9 reagents, the TALEN coding sequence was replaced with a plant codon-optimized Cas9 gene and the gRNAs were expressed from the AtU6 promoter (not shown). b–h Regeneration of tomato plants with targeted insertions. b Cotyledons of tomato cv. MicroTom after inoculation with Agrobacterium. c A recombinant explant 3 weeks after inoculation. Part of the developing callus accumulates anthocyanins due to the targeted promoter insertion and ANT1 overexpression. d Explants 5 weeks after inoculation. Small shoots begin to develop on the purple callus. e Multiple shoots growing from the purple callus 10–12 weeks after inoculation. f Plantlets develop roots 12–14 weeks after inoculation. g Plantlet transplanted to soil. h Dark purple coloration in flowers, fruit and foliage results from targeted promoter insertion. Flowers, fruit and mature plants are compared between wild type (WT) plants and those that have undergone GT. Scale bars = 1 cm
Fig. 3
PCR analysis of targeted insertions in 16 purple calli obtained from one transformation experiment. a Diagram of the ANT1 locus after gene targeting. Numbered arrows represent primers used in the study. b At the left junction, 11 of 16 purple calli gave the correct PCR product; 16 of 16 purple calli gave the correct product at the right junction. Products were obtained in all reactions with the PCR controls. Numbers represent purple calli corresponding to independent GT events. M 2-Log DNA ladder (New England Biolabs), WT wild type plant, NT no template control
Fig. 4
PCR and Southern blot analysis of GT events in pigmented plants. a Maps of the WT ANT1 locus, the ANT1 locus with a precise insertion, and an ANT1 locus that has sustained a one-sided GT event. Primers used for PCR are indicated by numbered arrows. b PCR results from 26 purple plants recovered from four independently derived purple calli (events 1, 2, 10 and 11). PCR products of the expected size were obtained from all plants at the right junction. PCR products of the expected size of the left junction were obtained in all plants from events 2 and 10 and all plants from event 1 except for plant 1.10. Of the plants regenerated from event 11, only plant 11.3 proved positive for the left junction. Viral replicons were not detected in any of the mature plants. Primers used for detecting viral replicons were the same as in Fig. S4 in Additional file 1. M 2-Log DNA ladder (New England BioLabs), WT wild type plant, C positive control for virus circularization (genomic DNA from tissue 8 weeks after inoculation with the viral GT vector). Plants selected for Southern blot analysis are marked by asterisks. c Southern blot analysis of NsiI-digested genomic DNA from purple plants 1.9, 11.1 and 2.5. The 4.4-kb band in plants 1.9 and 2.5 is the size expected for precise insertion by HR. Plant 11.1 showed an approximately 6.3-kb band, indicative of a one-sided GT event. The 2.5-kb WT band was detected in all plants, demonstrating that they are heterozygous for the targeted insertion. No other bands were detected in any of the tested GT plants, suggesting that random integration of the T-DNA did not occur
Fig. 5
PCR detection of one-sided and true GT events in plants derived from event 11. a Diagrams of true and one-sided GT events. Primers used for PCR are marked with numbered arrows. b PCR analysis confirmed one-sided GT events in plants 11.1, 11.2, 11.4 and 11.5 and a true GT event in plant 11.3. c Reconstruction of the one-sided GT event from plant 11.1. DNA sequence analysis revealed precise, HR-mediated repair on the right side. On the left side, before re-ligation of the broken chromosome, an additional 966 bp of sequence was copied from the GT vector and another 29 bp of unknown origin
Fig. 6
Transmission of the targeted insertion to the next generation. a Purple coloration is visible in the embryos within the seeds. b Scheme of the multiplexed PCR used to detect both WT and GT events in progeny of GT lines. Primers TC097F, ZY010F and TC210R (marked by arrows) were used in a single reaction. c A sample gel picture with products from PCR analysis of 30 T1 seedlings (gel pictures from PCR analysis of all 175 screened seedlings are provided in Fig. S12 in Additional file 1). All three possible genotypes were detected. Green arrow marks the WT products, the purple arrow the GT products, and red arrow the 1.0-kb band in the DNA ladder. The phenotype of each seedling is marked by P (purple) or G (green). M 2-Log DNA ladder (New England Biolabs), NT no template control. d–f Pictures of three of each homozygous WT (d) and heterozygous (e) and homozygous (f) GT T1 plants. The homozygous GT plants have reduced growth due to excessive accumulation of anthocyanins. Scale bars = 1 cm
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