| Autor: |
Gabsalilow, Lilia, Schierling, Benno, Friedhoff, Peter, Pingoud, Alfred, Wende, Wolfgang, Justus Liebig University Giessen |
| Jazyk: |
angličtina |
| Rok vydání: |
2013 |
| Předmět: |
|
| DOI: |
10.22029/jlupub-9056 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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