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Review
. 2013 Aug;70(16):2969-83.
doi: 10.1007/s00018-012-1204-1. Epub 2012 Nov 17.

Gene knockout and knockin by zinc-finger nucleases: current status and perspectives

J Hauschild-Quintern  1 , B Petersen, G J Cost, H Niemann
Affiliations
Review

Gene knockout and knockin by zinc-finger nucleases: current status and perspectives

J Hauschild-Quintern et al. Cell Mol Life Sci. 2013 Aug.

Abstract

Zinc-finger nucleases (ZFNs) are engineered site-specific DNA cleavage enzymes that may be designed to recognize long target sites and thus cut DNA with high specificity. ZFNs mediate permanent and targeted genetic alteration via induction of a double-strand break at a specific genomic site. Compared to conventional homology-based gene targeting, ZFNs can increase the targeting rate by up to 100,000-fold; gene disruption via mutagenic DNA repair is similarly efficient. The utility of ZFNs has been shown in many organisms, including insects, amphibians, plants, nematodes, and several mammals, including humans. This broad range of tractable species renders ZFNs a useful tool for improving the understanding of complex physiological systems, to produce transgenic animals, cell lines, and plants, and to treat human disease.

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Figures

Fig. 1
Fig. 1
a The zinc-finger molecule consists of one α-helix and two β sheets. The zinc ion (black ball) is bound by two cysteines of the β sheets and two histidines of the α-helix leading to a stabilization of the fold. Residues of the α-helix contact 3 bp of the DNA. b Four ZF molecules fused to a nuclease form a ZFN. Two ZFNs have to bind the targeted region in tail to tail direction to allow dimerization and cleavage of the FokI nuclease domain
Fig. 2
Fig. 2
After two ZFN molecules, each consisting of five zinc fingers, bind specifically to their target sequences, the nuclease domains dimerize and cut the DNA. Double-strand break repair by non homologous end joining (NHEJ) can induce mutations leading to a gene knockout by frame shift. If a donor DNA (grey) is added with homologous arms (white letters on grey background) to the targeted region, the sequence information between the homology arms of the donor DNA is copied into the genome and a gene knockin occurs
Fig. 3
Fig. 3
a ZFN targeting in pig cells: fetal or ear fibroblasts are obtained for cell culture. After expansion, cells are transfected (electroporation) with ZFN plasmids or mRNA. ZFN activity leads to gene targeting in cell culture and targeted cells are enriched (FACS, bead selection etc.) and injected into enucleated oocytes (SCNT). After fusion and activation, the embryos are transferred to synchronized sows and transgenic animals are born. b Zygotes at the pronuclear stage are obtained and injected with ZFN mRNA or ZFN plasmid DNA. If the ZFN cleaves both alleles in one-cell embryos, the targeted gene will be uniformly mutated throughout the embryo, leading to transgenic animals after transfer to a pseudopregnant female. If the ZFN does not cleave both alleles in the one-cell embryo, mosaic animals will result
Fig. 4
Fig. 4
a Cel-I assay (Surveyor nuclease assay) scheme: PCR products of wild-type DNA and mutated DNA of the ZFN-targeted locus are hybridized, allowing individual DNA strands to reassort. Incubation with the Surveyor nuclease cleaves the resulting base mismatches or loops. b The digestion products are electrophoresed; cleavage products are only seen when wild-type and mutant DNA are mixed (50 % PCR product, 25 % cleavage product each). When performed on ZFN-modified samples, the diversity of alleles generated by NHEJ allows the Cel-I assay to report the degree of gene modification
Fig. 5
Fig. 5
Mutated FokI nucleases only dimerize and cut (flash) the DNA if matching ZFNs (ZFN±) bind to its target DNA (obligate heterodimer). If the same ZFNs bind to an off-target site (homodimer formation: ZFN+/+or ZFN−/−) no cleavage occurs (modified from [33])
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References

    1. Rouet P, Smih F, Jasin M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci USA. 1994;91(13):6064–6068. doi: 10.1073/pnas.91.13.6064. - DOI - PMC - PubMed
    1. Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985;4(6):1609–1614. - PMC - PubMed
    1. Pabo CO, Peisach E, Grant RA. Design and selection of novel Cys2His2 zinc finger proteins. Annu Rev Biochem. 2001;70:313–340. doi: 10.1146/annurev.biochem.70.1.313. - DOI - PubMed
    1. Pavletich NP, Pabo CO. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science. 1991;252(5007):809–817. doi: 10.1126/science.2028256. - DOI - PubMed
    1. Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA. 1996;93(3):1156–1160. doi: 10.1073/pnas.93.3.1156. - DOI - PMC - PubMed

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