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Genome editing in plants via designed zinc finger nucleases.

Petolino JF - In Vitro Cell. Dev. Biol., Plant (2015)

Bottom Line: Targeted deletions of intervening DNA sequence can be obtained by ZFNs used to create concurrent DSBs.Site-specific transgene integration into ZFN-induced DSBs is possible via either NHEJ or HDR.Genome editing can be used to enhance our basic understanding of plant gene function as well as modify and improve crop plants.

View Article: PubMed Central - PubMed

Affiliation: Dow AgroSciences, 9330 Zionsville Rd., Indianapolis, IN USA.

ABSTRACT

The ability to create DNA double-strand breaks (DSBs) at specified genomic locations, which then stimulate the cell's naturally occurring DNA repair processes, has introduced intriguing possibilities for genetic modification. Zinc finger nucleases (ZFNs) are designed restriction enzymes consisting of a nonspecific cleavage domain fused to sequence-specific DNA binding domains. ZFN-mediated DSB formation at endogenous genomic loci followed by error-prone non-homologous end joining (NHEJ) repair can result in gene-specific mutations via nucleotide base pair insertions or deletions. Similarly, specific DNA sequence modifications can be made by providing donor DNA templates homologous to sequences flanking the cleavage site via homology-directed repair (HDR). Targeted deletions of intervening DNA sequence can be obtained by ZFNs used to create concurrent DSBs. Site-specific transgene integration into ZFN-induced DSBs is possible via either NHEJ or HDR. Genome editing can be used to enhance our basic understanding of plant gene function as well as modify and improve crop plants. As with conventional plant transformation technology, the efficiency of genome editing is absolutely dependent on the ability to initiate, maintain, and regenerate plant cell and tissue cultures.

No MeSH data available.


Related in: MedlinePlus

Gene deletion. Two concurrent zinc finger nuclease (ZFN)-mediated double-strand breaks (DSBs) can result in the loss of intervening sequences.
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Fig2: Gene deletion. Two concurrent zinc finger nuclease (ZFN)-mediated double-strand breaks (DSBs) can result in the loss of intervening sequences.

Mentions: In addition to small, NHEJ-induced mutations, ZFN-mediated cleavage can result in larger DNA sequence deletions (Fig. 2). A reporter construct containing a tandem repeat of 540 bp of partial, i.e., 3′/5′, GREEN FLUORESCENT PROTEIN (GFP) gene fragments with 2.8 kb of intervening heterologous DNA sequence containing a ZFN cleavage site was stably integrated into tobacco (Cai et al.2009). Subsequent expression of the corresponding ZFN gene resulted in targeted DSB formation, recombination between the GFP gene fragments, and deletion of the intervening 2.8-kb sequence. Deletion of a 4.3-kb integrated GUS gene sequence flanked by ZFN cleavage sites was observed in 35% of the F1 progenies when crossed to a ZFN-expressing plant (Petolino et al.2010). Even larger deletions (e.g., 55 kb) were observed following nuclease cleavage within tandem gene clusters (Voytas 2013). These results provide proof of concept for a potentially powerful means of creating targeted genome sequence deletions.Figure 2.


Genome editing in plants via designed zinc finger nucleases.

Petolino JF - In Vitro Cell. Dev. Biol., Plant (2015)

Gene deletion. Two concurrent zinc finger nuclease (ZFN)-mediated double-strand breaks (DSBs) can result in the loss of intervening sequences.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4352198&req=5

Fig2: Gene deletion. Two concurrent zinc finger nuclease (ZFN)-mediated double-strand breaks (DSBs) can result in the loss of intervening sequences.
Mentions: In addition to small, NHEJ-induced mutations, ZFN-mediated cleavage can result in larger DNA sequence deletions (Fig. 2). A reporter construct containing a tandem repeat of 540 bp of partial, i.e., 3′/5′, GREEN FLUORESCENT PROTEIN (GFP) gene fragments with 2.8 kb of intervening heterologous DNA sequence containing a ZFN cleavage site was stably integrated into tobacco (Cai et al.2009). Subsequent expression of the corresponding ZFN gene resulted in targeted DSB formation, recombination between the GFP gene fragments, and deletion of the intervening 2.8-kb sequence. Deletion of a 4.3-kb integrated GUS gene sequence flanked by ZFN cleavage sites was observed in 35% of the F1 progenies when crossed to a ZFN-expressing plant (Petolino et al.2010). Even larger deletions (e.g., 55 kb) were observed following nuclease cleavage within tandem gene clusters (Voytas 2013). These results provide proof of concept for a potentially powerful means of creating targeted genome sequence deletions.Figure 2.

Bottom Line: Targeted deletions of intervening DNA sequence can be obtained by ZFNs used to create concurrent DSBs.Site-specific transgene integration into ZFN-induced DSBs is possible via either NHEJ or HDR.Genome editing can be used to enhance our basic understanding of plant gene function as well as modify and improve crop plants.

View Article: PubMed Central - PubMed

Affiliation: Dow AgroSciences, 9330 Zionsville Rd., Indianapolis, IN USA.

ABSTRACT

The ability to create DNA double-strand breaks (DSBs) at specified genomic locations, which then stimulate the cell's naturally occurring DNA repair processes, has introduced intriguing possibilities for genetic modification. Zinc finger nucleases (ZFNs) are designed restriction enzymes consisting of a nonspecific cleavage domain fused to sequence-specific DNA binding domains. ZFN-mediated DSB formation at endogenous genomic loci followed by error-prone non-homologous end joining (NHEJ) repair can result in gene-specific mutations via nucleotide base pair insertions or deletions. Similarly, specific DNA sequence modifications can be made by providing donor DNA templates homologous to sequences flanking the cleavage site via homology-directed repair (HDR). Targeted deletions of intervening DNA sequence can be obtained by ZFNs used to create concurrent DSBs. Site-specific transgene integration into ZFN-induced DSBs is possible via either NHEJ or HDR. Genome editing can be used to enhance our basic understanding of plant gene function as well as modify and improve crop plants. As with conventional plant transformation technology, the efficiency of genome editing is absolutely dependent on the ability to initiate, maintain, and regenerate plant cell and tissue cultures.

No MeSH data available.


Related in: MedlinePlus