title: Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion.
abstract: Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The generation of obligate heterodimeric ZFNs was shown to significantly reduce ZFN-associated cytotoxicity in single-site genome editing strategies. To further expand the application range of ZFNs, we employed a combination of in silico protein modeling, in vitro cleavage assays, and in vivo recombination assays to identify autonomous ZFN pairs that lack cross-reactivity between each other. In the context of ZFNs designed to recognize two adjacent sites in the human HOXB13 locus, we demonstrate that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼ 10% of alleles. Notably, the autonomous ZFN pair induced a targeted chromosomal deletion with the same efficacy as previously published obligate heterodimeric ZFNs but with significantly less toxicity. These results demonstrate that autonomous ZFNs will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct ZFN pairs.
authors: Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors: Cem
authors: C
authors: Söllü
authors: Kaweh
authors: K
authors: Pars
authors: Tatjana I
authors: TI
authors: Cornu
authors: Stacey
authors: S
authors: Thibodeau-Beganny
authors: Morgan L
authors: ML
authors: Maeder
authors: J Keith
authors: JK
authors: Joung
authors: Regine
authors: R
authors: Heilbronn
authors: Toni
authors: T
authors: Cathomen
mesh_terms: D020134:Catalytic Domain; D002460:Cell Line; D002872:Chromosome Deletion; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D005818:Genetic Engineering; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; C484414:HOXB13 protein, human; D018398:Homeodomain Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific