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Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats

Gabsalilow, Lilia ; Schierling, Benno ; Friedhoff, Peter ; Pingoud, Alfred ; Wende, Wolfgang


Originalveröffentlichung: (2013) Nucleic Acids Research; doi:10.1093/nar/gkt080
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URN: urn:nbn:de:hebis:26-opus-92283
URL: http://geb.uni-giessen.de/geb/volltexte/2013/9228/

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Sammlung: Open Access - Publikationsfonds
Universität Justus-Liebig-Universität Gießen
Institut: Institute for Biochemistry
Fachgebiet: Biochemie (FB 08)
DDC-Sachgruppe: Biowissenschaften, Biologie
Dokumentart: Aufsatz
Sprache: Englisch
Erstellungsjahr: 2013
Publikationsdatum: 04.03.2013
Kurzfassung auf Englisch: Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by ‘nickases’ that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE–MutH is a programmable nickase.
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