Meganucleases and Their Biomedical Applications


Homing endonucleases and zinc‐finger nucleases are double‐stranded meganucleases that target large deoxyribonucleic acid (DNA) recognition sites, a feature that means only a few such sites are likely to be present in a mammalian‐sized genome. Several homing endonucleases have been used as templates to engineer tools that cleave DNA sequences other than their original wild‐type targets. Indeed, zinc‐finger nucleases have been designed to recognise specific genes. These custom meganucleases can be used to stimulate double‐strand break homologous recombination in cells, thereby inducing the repair of defective genes with very low toxicity levels. The use of tailored meganucleases opens up new possibilities for gene therapy in patients with monogenic diseases that can be treated ex vivo. This review provides an overview of recent advances in this field.

Key Concepts:

  • Meganucleases are double‐stranded DNAses that target large recognition sites.

  • Meganucleases promote efficient gene targeting through double‐strand‐break‐induced homologous recombination.

  • Homologous recombination and nonhomologous end joining are the two pathways that can be used to repair DNA double‐strand breaks.

  • Meganucleases encompass homing endonucleases and zinc‐finger nucleases.

  • Meganucleases are ideal scaffolds to engineer enzymes that accurately cleave DNA and induce recombination.

  • Meganucleases can recognise unique sites of cleavage within a genome, permitting their use for therapeutic purposes in human diseases.

  • X‐ray diffraction of crystallised proteins allows us to understand the relationship between the structure and function of biomolecules.

Keywords: homing endonucleases; zinc‐finger nucleases; protein engineering; monogenic diseases; double‐strand break; single‐strand break; DNA repair; gene therapy; protein structure

Figure 1.

Cellular DNA damage can be repaired by the DSB or SSB repair pathway. Cleavage of the intended target gene can lead to disruption of its coding sequence by inaccurate repair via NHEJ or, when a donor DNA (orange bar) is introduced along with an HE or ZFN, it can be incorporated at the target by HR. SSBs are repaired by the base excision repair pathway.

Figure 2.

Crystallographic structures of representative members of the five HE families. (a) LAGLIDADG family: monomeric I‐DmoI (2VS7) and homodimeric I‐CreI (1G9Y). (b) PD‐D/E‐XK family: the tetrameric I‐SspI (2OST). (c) His‐Cys Box family: the homodimeriac I‐PpoI (1CYQ). (d) GIY‐YIG family: I‐TevI catalytic (1LN0) and DNA‐binding domains (1I3J). (e) HNH family: the monomeric I‐HmuI (1U3E). (f) Engineered heterodimeric variants AmeI and Ini based on the I‐CreI template. (a)–(e) The enzymes are shown as a cartoon representation with the bound target site in stick representation. Catalytic ions are shown as yellow spheres and structural zinc ions are shown as orange spheres.

Figure 3.

The zinc‐finger motif and DNA recognition. (a) Ribbon diagram of a Cys2His2 zinc finger, including the two cysteine side chains (grey) and two histidine side chains (blue) that coordinate the zinc ion (green sphere: PDB code: 1AAY). Orange side chains correspond to residues that recognise specific DNA bases and that make contacts in the major groove of the DNA. (b) Crystallographic structure (1P47) of a homodimeric ZF composed of three fingers. The colour code is the same as in (a). (c) corresponds to (b) representation 90° left‐rotated.

Figure 4.

ZFN gene targeting in human lymphocytes. (a) Representation of a ZFN recognising the human IL‐2Rγ gene target. (b) Mechanism of SCID repair by homologous recombination in human cells. IL‐2Rγ(−) shows a SCID mutation, and the repair is carried out by cells cotransfected with both ZFN and a nonmutated DNA fragment of the wild‐type IL‐2Rγ gene.



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Further Reading

Chevalier BS, Kortemme T, Chadsey MS et al. (2002) Design, activity, and structure of a highly specific artificial endonuclease. Molecular Cell 10(4): 895–905.

Epinat JC, Arnould S, Chames P et al. (2003) A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Research 31(11): 2952–2962.

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Perez EE, Wang J, Miller JC et al. (2008) Establishment of HIV‐1 resistance in CD4+ T cells by genome editing using zinc‐finger nucleases. Nature Biotechnology 26(7): 808–816.

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Molina, Rafael, Montoya, Guillermo, and Prieto, Jesús(Jan 2011) Meganucleases and Their Biomedical Applications. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0023179]