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Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley.

Budhagatapalli N, Rutten T, Gurushidze M, Kumlehn J, Hensel G - G3 (Bethesda) (2015)

Bottom Line: Transcription activator-like effector nucleases open up new opportunities for targeted mutagenesis in eukaryotic genomes.As a result of co-bombardment of leaf epidermis, we detected yellow fluorescent protein accumulation in about three of 100 mutated cells.The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site.

View Article: PubMed Central - PubMed

Affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Stadt Seeland/OT Gatersleben, Germany.

No MeSH data available.


Polymerase chain reaction (PCR) analysis and DNA-sequencing confirm gfp-to-yfp conversion within the host genome. (A) PCR products amplified from genomic DNA extracted from leaves of lines 319L (upper gel) and 335R (lower gel) without bombardment are shown in lanes 1−4, of those bombarded with the linearized truncated yfp* fragment alone in lanes 5−8, and of those bombarded with both the linearized truncated yfp* fragment and the complementary TALEN unit (pGH400 for right-hand, pGH297 for the left-hand unit) in lanes 9−14. pGFP was the negative control [plasmid carrying gfp transfer DNA (T-DNA)] and pYFP positive control (plasmid carrying yfp T-DNA). (B) Verification of allele conversion via sequencing; presented are sequences of donor plant (gDNA), the truncated yfp* fragment used as repair template, the genomic target sequence after homology-directed repair of TALEN-mediated DSBs, forward and reverse primers used for DNA-amplification from samples of different leaves of lines 319L (left-hand TALEN unit) and 335R (right-hand TALEN unit) 24 hr after bombardment as well as sequencing data providing evidence of gfp-to-yfp conversion. The sequences recovered from all other independent leaves were identical to those three per plant line shown here. Nucleotides different in the yfp* sequence from those of gfp are printed in blue. The binding site of the right TALEN unit is underlined. An asterisk indicates an altered target sequence after leaf bombardment in lines 339L and 335R. The dotted line indicates the end of the repair template. XFP, green or yellow fluorescent protein; Thr/Tyr, amino acid change responsible for GFP-to-YFP conversion; NOST, A. tumefaciens NOPALINE SYNTHASE transcriptional termination sequence; 35SP, CaMV 35S promoter.
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fig3: Polymerase chain reaction (PCR) analysis and DNA-sequencing confirm gfp-to-yfp conversion within the host genome. (A) PCR products amplified from genomic DNA extracted from leaves of lines 319L (upper gel) and 335R (lower gel) without bombardment are shown in lanes 1−4, of those bombarded with the linearized truncated yfp* fragment alone in lanes 5−8, and of those bombarded with both the linearized truncated yfp* fragment and the complementary TALEN unit (pGH400 for right-hand, pGH297 for the left-hand unit) in lanes 9−14. pGFP was the negative control [plasmid carrying gfp transfer DNA (T-DNA)] and pYFP positive control (plasmid carrying yfp T-DNA). (B) Verification of allele conversion via sequencing; presented are sequences of donor plant (gDNA), the truncated yfp* fragment used as repair template, the genomic target sequence after homology-directed repair of TALEN-mediated DSBs, forward and reverse primers used for DNA-amplification from samples of different leaves of lines 319L (left-hand TALEN unit) and 335R (right-hand TALEN unit) 24 hr after bombardment as well as sequencing data providing evidence of gfp-to-yfp conversion. The sequences recovered from all other independent leaves were identical to those three per plant line shown here. Nucleotides different in the yfp* sequence from those of gfp are printed in blue. The binding site of the right TALEN unit is underlined. An asterisk indicates an altered target sequence after leaf bombardment in lines 339L and 335R. The dotted line indicates the end of the repair template. XFP, green or yellow fluorescent protein; Thr/Tyr, amino acid change responsible for GFP-to-YFP conversion; NOST, A. tumefaciens NOPALINE SYNTHASE transcriptional termination sequence; 35SP, CaMV 35S promoter.

Mentions: Because somatic cells are diploid and therefore carry two copies of each gene, the loss of GFP activity would require the loss-of-function of both copies. Therefore, mutation efficiency is easily underestimated. A number of the yfp-expressing cells coexpressed gfp, but others clearly showed solely YFP activity (Figure 2, A−C, left cell, Figure S3, C-E). This fact emphasizes how fast the process of DNA repair after TALEN-mediated DSB induction can take place. In contrast, it was somehow surprising that in yellow fluorescent cells 1 d after bombardment, the GFP protein was already depleted to below the detection limit. To confirm that the genomic gfp sequence had been converted into the yfp, PCR was conducted using primers (Table S1) specific for yfp and the 35S promoter driving the selectable marker gene (HPT), the latter expression unit being present within the stably inserted transfer DNA that also carries the gfp gene. Thus, an amplicon could only be generated if a TALEN-induced DSB had been processed by HDR, based on the yfp fragment supplied in the form of the repair template (Figure 3A). Sequencing of the fragments recovered from the amplicons unambiguously confirmed that the desired conversion had occurred in the genomic DNA (Figure 3B). The principle of gfp-to-yfp conversion introduced in this study might be instrumental in future studies on genome editing. For instance, one could optimize various properties of customizable endonucleases like TALEN spacer length, target motif composition, as well as editing-related features such as the size of the repair template, length of homologous regions, or overexpression of genes involved in the HDR process. A challenge that remains to be overcome before the principle demonstrated here at the cellular level can be extended to the regeneration of plants carrying a precisely predicted modification is that such genome editing events are not typically associated with any selective advantage. As a result, alternatives to the conventional use of selective agents (such as herbicide or antibiotic resistance) will need to be elaborated. However, for the moment, what has been demonstrated is the proof of concept that any plant gene can be modified in a predictable way, thereby opening the door to redesigning native gene sequences to modify their function or to reverse mutations in a directed way.


Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley.

Budhagatapalli N, Rutten T, Gurushidze M, Kumlehn J, Hensel G - G3 (Bethesda) (2015)

Polymerase chain reaction (PCR) analysis and DNA-sequencing confirm gfp-to-yfp conversion within the host genome. (A) PCR products amplified from genomic DNA extracted from leaves of lines 319L (upper gel) and 335R (lower gel) without bombardment are shown in lanes 1−4, of those bombarded with the linearized truncated yfp* fragment alone in lanes 5−8, and of those bombarded with both the linearized truncated yfp* fragment and the complementary TALEN unit (pGH400 for right-hand, pGH297 for the left-hand unit) in lanes 9−14. pGFP was the negative control [plasmid carrying gfp transfer DNA (T-DNA)] and pYFP positive control (plasmid carrying yfp T-DNA). (B) Verification of allele conversion via sequencing; presented are sequences of donor plant (gDNA), the truncated yfp* fragment used as repair template, the genomic target sequence after homology-directed repair of TALEN-mediated DSBs, forward and reverse primers used for DNA-amplification from samples of different leaves of lines 319L (left-hand TALEN unit) and 335R (right-hand TALEN unit) 24 hr after bombardment as well as sequencing data providing evidence of gfp-to-yfp conversion. The sequences recovered from all other independent leaves were identical to those three per plant line shown here. Nucleotides different in the yfp* sequence from those of gfp are printed in blue. The binding site of the right TALEN unit is underlined. An asterisk indicates an altered target sequence after leaf bombardment in lines 339L and 335R. The dotted line indicates the end of the repair template. XFP, green or yellow fluorescent protein; Thr/Tyr, amino acid change responsible for GFP-to-YFP conversion; NOST, A. tumefaciens NOPALINE SYNTHASE transcriptional termination sequence; 35SP, CaMV 35S promoter.
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fig3: Polymerase chain reaction (PCR) analysis and DNA-sequencing confirm gfp-to-yfp conversion within the host genome. (A) PCR products amplified from genomic DNA extracted from leaves of lines 319L (upper gel) and 335R (lower gel) without bombardment are shown in lanes 1−4, of those bombarded with the linearized truncated yfp* fragment alone in lanes 5−8, and of those bombarded with both the linearized truncated yfp* fragment and the complementary TALEN unit (pGH400 for right-hand, pGH297 for the left-hand unit) in lanes 9−14. pGFP was the negative control [plasmid carrying gfp transfer DNA (T-DNA)] and pYFP positive control (plasmid carrying yfp T-DNA). (B) Verification of allele conversion via sequencing; presented are sequences of donor plant (gDNA), the truncated yfp* fragment used as repair template, the genomic target sequence after homology-directed repair of TALEN-mediated DSBs, forward and reverse primers used for DNA-amplification from samples of different leaves of lines 319L (left-hand TALEN unit) and 335R (right-hand TALEN unit) 24 hr after bombardment as well as sequencing data providing evidence of gfp-to-yfp conversion. The sequences recovered from all other independent leaves were identical to those three per plant line shown here. Nucleotides different in the yfp* sequence from those of gfp are printed in blue. The binding site of the right TALEN unit is underlined. An asterisk indicates an altered target sequence after leaf bombardment in lines 339L and 335R. The dotted line indicates the end of the repair template. XFP, green or yellow fluorescent protein; Thr/Tyr, amino acid change responsible for GFP-to-YFP conversion; NOST, A. tumefaciens NOPALINE SYNTHASE transcriptional termination sequence; 35SP, CaMV 35S promoter.
Mentions: Because somatic cells are diploid and therefore carry two copies of each gene, the loss of GFP activity would require the loss-of-function of both copies. Therefore, mutation efficiency is easily underestimated. A number of the yfp-expressing cells coexpressed gfp, but others clearly showed solely YFP activity (Figure 2, A−C, left cell, Figure S3, C-E). This fact emphasizes how fast the process of DNA repair after TALEN-mediated DSB induction can take place. In contrast, it was somehow surprising that in yellow fluorescent cells 1 d after bombardment, the GFP protein was already depleted to below the detection limit. To confirm that the genomic gfp sequence had been converted into the yfp, PCR was conducted using primers (Table S1) specific for yfp and the 35S promoter driving the selectable marker gene (HPT), the latter expression unit being present within the stably inserted transfer DNA that also carries the gfp gene. Thus, an amplicon could only be generated if a TALEN-induced DSB had been processed by HDR, based on the yfp fragment supplied in the form of the repair template (Figure 3A). Sequencing of the fragments recovered from the amplicons unambiguously confirmed that the desired conversion had occurred in the genomic DNA (Figure 3B). The principle of gfp-to-yfp conversion introduced in this study might be instrumental in future studies on genome editing. For instance, one could optimize various properties of customizable endonucleases like TALEN spacer length, target motif composition, as well as editing-related features such as the size of the repair template, length of homologous regions, or overexpression of genes involved in the HDR process. A challenge that remains to be overcome before the principle demonstrated here at the cellular level can be extended to the regeneration of plants carrying a precisely predicted modification is that such genome editing events are not typically associated with any selective advantage. As a result, alternatives to the conventional use of selective agents (such as herbicide or antibiotic resistance) will need to be elaborated. However, for the moment, what has been demonstrated is the proof of concept that any plant gene can be modified in a predictable way, thereby opening the door to redesigning native gene sequences to modify their function or to reverse mutations in a directed way.

Bottom Line: Transcription activator-like effector nucleases open up new opportunities for targeted mutagenesis in eukaryotic genomes.As a result of co-bombardment of leaf epidermis, we detected yellow fluorescent protein accumulation in about three of 100 mutated cells.The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site.

View Article: PubMed Central - PubMed

Affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Stadt Seeland/OT Gatersleben, Germany.

No MeSH data available.