Journal Article

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

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

in Nucleic Acids Research

Volume 41, issue 7, pages e83-e83
Published in print April 2013 | ISSN: 0305-1048
Published online February 2013 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gkt080

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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.

Journal Article.  6141 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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