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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

Targeted genome modification via double-strand break (DSB) repair. Zinc finger nucleases (ZFNs) bind to a target sequence, thereby dimerizing FokI nuclease. The DSB generated by ZFN cleavage induces DNA repair processes. In the absence of donor template DNA, error-prone non-homologous end joining (NHEJ) can result in ‘targeted mutagenesis’ (left). In the presence of homologous sequences, homology-directed repair (HDR) can result in ‘gene editing’ (center) or ‘site-specific integration’ (right).
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Fig1: Targeted genome modification via double-strand break (DSB) repair. Zinc finger nucleases (ZFNs) bind to a target sequence, thereby dimerizing FokI nuclease. The DSB generated by ZFN cleavage induces DNA repair processes. In the absence of donor template DNA, error-prone non-homologous end joining (NHEJ) can result in ‘targeted mutagenesis’ (left). In the presence of homologous sequences, homology-directed repair (HDR) can result in ‘gene editing’ (center) or ‘site-specific integration’ (right).

Mentions: ZFNs consist of zinc finger protein domains, capable of sequence-specific DNA binding, fused to a nuclease domain for DNA cleavage (Fig. 1). DNA binding is the result of a tethered array of 4–6 zinc finger protein domains that each recognize approximately 3 bp of DNA. Although more-or-less modular with respect to binding specificity, there are considerable context effects, i.e., interactions with neighboring domains, which make their binding more specific but their design somewhat challenging (Urnov et al.2010). They are most effectively assembled from an archive of two-finger modules that each recognize specific 6-bp DNA sequences whereby domain junctions within each module are optimized for sequence recognition (Moore et al.2001). For DNA cleavage, the catalytic domain of the type II restriction enzyme FokI has been used (Kim et al.1996). A critical property of the catalytic domain is that it must dimerize to cleave DNA, so two adjacent ZFN pairs must orient themselves with appropriate spacing at the target site (Fig. 1). Although a somewhat larger gene product needs to be expressed, the longer recognition sequences (24–36 bp) required for binding result in a higher level of specificity. In addition, FokI variants requiring heterodimerization have been developed, thereby further enhancing sequence specificity and reducing off-site cleavage (Miller et al.2007).Figure 1.


Genome editing in plants via designed zinc finger nucleases.

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

Targeted genome modification via double-strand break (DSB) repair. Zinc finger nucleases (ZFNs) bind to a target sequence, thereby dimerizing FokI nuclease. The DSB generated by ZFN cleavage induces DNA repair processes. In the absence of donor template DNA, error-prone non-homologous end joining (NHEJ) can result in ‘targeted mutagenesis’ (left). In the presence of homologous sequences, homology-directed repair (HDR) can result in ‘gene editing’ (center) or ‘site-specific integration’ (right).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Targeted genome modification via double-strand break (DSB) repair. Zinc finger nucleases (ZFNs) bind to a target sequence, thereby dimerizing FokI nuclease. The DSB generated by ZFN cleavage induces DNA repair processes. In the absence of donor template DNA, error-prone non-homologous end joining (NHEJ) can result in ‘targeted mutagenesis’ (left). In the presence of homologous sequences, homology-directed repair (HDR) can result in ‘gene editing’ (center) or ‘site-specific integration’ (right).
Mentions: ZFNs consist of zinc finger protein domains, capable of sequence-specific DNA binding, fused to a nuclease domain for DNA cleavage (Fig. 1). DNA binding is the result of a tethered array of 4–6 zinc finger protein domains that each recognize approximately 3 bp of DNA. Although more-or-less modular with respect to binding specificity, there are considerable context effects, i.e., interactions with neighboring domains, which make their binding more specific but their design somewhat challenging (Urnov et al.2010). They are most effectively assembled from an archive of two-finger modules that each recognize specific 6-bp DNA sequences whereby domain junctions within each module are optimized for sequence recognition (Moore et al.2001). For DNA cleavage, the catalytic domain of the type II restriction enzyme FokI has been used (Kim et al.1996). A critical property of the catalytic domain is that it must dimerize to cleave DNA, so two adjacent ZFN pairs must orient themselves with appropriate spacing at the target site (Fig. 1). Although a somewhat larger gene product needs to be expressed, the longer recognition sequences (24–36 bp) required for binding result in a higher level of specificity. In addition, FokI variants requiring heterodimerization have been developed, thereby further enhancing sequence specificity and reducing off-site cleavage (Miller et al.2007).Figure 1.

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