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

Institute for Biochemistry, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 02/2013; 41(7). DOI: 10.1093/nar/gkt080
Source: PubMed

ABSTRACT 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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    • "To avoid such effects, several groups have inactivated one of the nuclease active sites to convert meganucleases [117] [118], ZFNs [119] and most recently also Cas9 [17] into nickases that make only singlestrand breaks (SSBs). Another approach consisted in fusing the nicking activity of the natural occurring DNA mismatch repair endonuclease MutH with the DNA-binding domain of meganucleases or TALE protein [120]. Since SSBs are not substrates for NHEJ but are repaired either by seamless ligation or high-fidelity HR, the ratio HR:NHEJ is improved and off-target effects are reduced. "
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Peter Friedhoff