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High-frequency, precise modification of the tomato genome.

Čermák T, Baltes NJ, Čegan R, Zhang Y, Voytas DF - Genome Biol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, 55455, USA. tcermak@umn.edu.

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.

No MeSH data available.


Related in: MedlinePlus

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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Fig6: 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

Mentions: To test whether the targeted DNA insertions were heritable, we analyzed progeny of 24 plants regenerated from events 1, 2, 11 and 14 (Fig. 6 and Table 2). A total of 123 T1 seedlings showed the characteristic purple color, which was already visible at the embryo stage within the seed (Fig. 6a). PCR analysis confirmed that 100 of these seedlings (57.1 %) were heterozygous and 23 (13.1 %) were homozygous for the promoter insertion; the other 52 green seedlings were WT (Table 2; Figure S12 in Additional file 1). Collectively, 70.2 % of the progeny were purple and 29.7 % were green. These data are consistent with the T0 plants being heterozygous for the targeted modification: all but 5 of the 24 plants segregated green progeny, and of these five, only a few seeds were produced. The number of plants carrying the modified ANT1 allele in the homozygous state was slightly lower than the expected 1:2:1 segregation frequency. This could be caused by a growth inhibitory effect resulting from excessive accumulation of anthocyanins [8]. Growth inhibition was observed to be much stronger in the homozygous plants (Fig. 6f) than the heterozygotes (Fig. 6e), the latter of which grew comparably to WT (Fig. 6d). It is possible that seed viability/germination is also affected by the excess pigments, which would result in the observed underrepresentation of homozygous ANT1 overexpressing plants in the T1 progeny. Furthermore, this inhibitory effect might also have been the reason why homozygous plants were not recovered in the T0 generation. We indeed observed that many potentially homozygous purple calli did not regenerate shoots; however, due to the small size of the calli, we could not test whether they were homozygous or not, as we could never be 100 % sure that only purple tissue was excised without a few WT cells from the surrounding, non-transformed tissue, which would subsequently cause all the samples to look like heterozygotes when analyzed by PCR. Therefore, to test this hypothesis, we conducted an experiment in which we directly tested the regenerative capacity of homo- and heterozygous tissue derived from the cotyledons of PCR-genotyped T1 seedlings. We did not find any difference between the samples in terms of callus and shoot induction (Figure S13 in Additional file 1). Thus, it remains unclear why homozygous plants were not regenerated in the T0 generation, and it may simply be that the frequency of HR is too low to recover bi-allelic events in the small number of plants generated. Importantly, we did demonstrate that plants homozygous for the insertion can be recovered in the T1 generation, and these results collectively demonstrate that our approach generates heritable genomic modifications.Fig. 6


High-frequency, precise modification of the tomato genome.

Čermák T, Baltes NJ, Čegan R, Zhang Y, Voytas DF - Genome Biol. (2015)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: 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
Mentions: To test whether the targeted DNA insertions were heritable, we analyzed progeny of 24 plants regenerated from events 1, 2, 11 and 14 (Fig. 6 and Table 2). A total of 123 T1 seedlings showed the characteristic purple color, which was already visible at the embryo stage within the seed (Fig. 6a). PCR analysis confirmed that 100 of these seedlings (57.1 %) were heterozygous and 23 (13.1 %) were homozygous for the promoter insertion; the other 52 green seedlings were WT (Table 2; Figure S12 in Additional file 1). Collectively, 70.2 % of the progeny were purple and 29.7 % were green. These data are consistent with the T0 plants being heterozygous for the targeted modification: all but 5 of the 24 plants segregated green progeny, and of these five, only a few seeds were produced. The number of plants carrying the modified ANT1 allele in the homozygous state was slightly lower than the expected 1:2:1 segregation frequency. This could be caused by a growth inhibitory effect resulting from excessive accumulation of anthocyanins [8]. Growth inhibition was observed to be much stronger in the homozygous plants (Fig. 6f) than the heterozygotes (Fig. 6e), the latter of which grew comparably to WT (Fig. 6d). It is possible that seed viability/germination is also affected by the excess pigments, which would result in the observed underrepresentation of homozygous ANT1 overexpressing plants in the T1 progeny. Furthermore, this inhibitory effect might also have been the reason why homozygous plants were not recovered in the T0 generation. We indeed observed that many potentially homozygous purple calli did not regenerate shoots; however, due to the small size of the calli, we could not test whether they were homozygous or not, as we could never be 100 % sure that only purple tissue was excised without a few WT cells from the surrounding, non-transformed tissue, which would subsequently cause all the samples to look like heterozygotes when analyzed by PCR. Therefore, to test this hypothesis, we conducted an experiment in which we directly tested the regenerative capacity of homo- and heterozygous tissue derived from the cotyledons of PCR-genotyped T1 seedlings. We did not find any difference between the samples in terms of callus and shoot induction (Figure S13 in Additional file 1). Thus, it remains unclear why homozygous plants were not regenerated in the T0 generation, and it may simply be that the frequency of HR is too low to recover bi-allelic events in the small number of plants generated. Importantly, we did demonstrate that plants homozygous for the insertion can be recovered in the T1 generation, and these results collectively demonstrate that our approach generates heritable genomic modifications.Fig. 6

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, 55455, USA. tcermak@umn.edu.

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.

No MeSH data available.


Related in: MedlinePlus