Molecular scissors for in situ cellular repair

Abstract

The engineering of protein–DNA interactions in different protein scaffolds may provide “toolkits” to modify the genome. Homing endonucleases are powerful tools for genome manipulation through homologous recombination, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. Therefore, the combination of a precise “cutter” with the presence of a natural or modified homologous DNA donor provides a potentially useful means to modify the genome. However, the basis of protein–DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. The engineering of homing endonucleases and alternative scaffolds, such as zinc fingers or transcription activator-like effector domains, has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Customized homing endonucleases targeting selected human genes can excise or correct regions of genes implicated in monogenic diseases, thereby representing important tools for intervention in eukaryotic genomes.

Keywords::

• Homing endonucleases
• gene targeting
• homologous recombination
• DNA double-strand break
• non-homologous end joining
• chimeric proteins
• nicking enzymes

Acknowledgements

We thank the Swiss Light Source and the European Synchrotron Radiation Facility beamline staff for their support.

Declaration of interest

Funding was obtained from Ministerio de Ciencia e Innovación (MICINN), through grants JCI-2011-09308 to R.M. and BFU2008-01344/BMC, BFU2011–23815/BMC and CSD2006–20642 to G.M., and from the Comunidad Autónoma de Madrid, grant CAM-P2006/Gen-0166 to G.M.

Editor: Michael M. Cox