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


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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    • "In the past few years, zinc finger nickases as fusions of zinc finger protein and FokI cleavage domains have been constructed to introduce sequence-specific nicks in genomic DNA for gene targeting (gene addition and gene deletion) (14,15). A nicking domain (ND) variant of a DNA repair enzyme (MutH) has been fused to a TAL effector protein to create a rare nicking enzyme suitable for gene targeting (16). However, no robust 5mC-specific nickases have been found in nature despite extensive enzyme screening in the past 30 years (see REBASE) (17). "
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    ABSTRACT: N.ϕGamma is a strand-specific and site-specific DNA nicking enzyme (YCG↓GT or AC↑CGR). Here we describe the isolation of single and double mutants of N.ϕGamma with attenuated activity. The nicking domains (NDs) of E59A and 11 double mutants were fused to the 5mCG-binding domain of MBD2 and generated fusion enzymes that preferentially nick 5mCG-modified DNA. The CG dinucleotide can be modified by C5 methyltransferases (MTases) such as M.SssI, M.HhaI or M.HpaII to create composite sites AC↑YGG N(8–15) 5mCG. We also constructed a fusion enzyme 2xMBD2-ND(N.BceSVIII) targeting more frequent composite sites AS↑YS N(5–12) 5mCG in Mn2+ buffer. 5mCG-dependent nicking requires special digestion conditions in high salt (0.3 M KCl) or in Ni2+ buffer. The fusion enzyme can be used to nick and label 5mCG-modified plasmid and genomic DNAs with fluorescently labeled Cy3-dUTP and potentially be useful for diagnostic applications, DNA sequencing and optical mapping of epigenetic markers. The importance of the predicted catalytic residues D89, H90, N106 and H115 in N.ϕGamma was confirmed by mutagenesis. We found that the wild-type enzyme N.ϕGamma prefers to nick 5mCG-modified DNA in Ni2+ buffer even though the nicking activity is sub-optimal compared to the activity in Mg2+ buffer.
    Nucleic Acids Research 03/2014; 42(9). DOI:10.1093/nar/gku192 · 9.11 Impact Factor
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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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    ABSTRACT: Nuclease-based gene targeting (NBGT) represents a significant breakthrough in targeted genome editing since it is applicable from single-celled protozoa to human, including a number of species of economic importance. Along with the fast progress in NBGT and the increasing availability of customized nucleases, more data are available about off-target effects associated with the use of this approach. We discuss how NBGT may offer a new perspective for genetic modification, we address some aspects crucial for a safety improvement of the corresponding techniques and we also briefly relate the use of NBGT applications and products to the regulatory oversight.
    New Biotechnology 01/2014; 31(1):18-27. DOI:10.1016/j.nbt.2013.07.001 · 2.90 Impact Factor
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    • "Of course, highly specific nucleases, regardless of which DNA-cleavage module they contain, need to be tested in vivo for their specificity which means for off-target cleavage, as it was done for ZFNs in a genome-wide analysis [47,48]. Finally, we have now expanded the “tool box” for gene targeting by a double-strand specific TALE-PvuII nuclease, which might be the nuclease of choice for gene disruption, whereas a nicking nuclease, such as TALE-MutH [57], might be more useful for gene replacement where one wants to avoid non-homologous end joining (NHEJ). "
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    ABSTRACT: Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.
    PLoS ONE 12/2013; 8(12):e82539. DOI:10.1371/journal.pone.0082539 · 3.23 Impact Factor
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