Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering.

Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein–DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein–DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein–DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein–DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein–DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.

Over the past 15 years, site-directed genome modi fi cation has proven to be an ef fi cient and robust approach. Meganucleases, sequence-speci fi c endonucleases with long recognition sites, represent one of several tools described in this book that can be used for this purpose. This chapter will review the early stages of the technology (with the fi rst strand break-induced gene targeting with I-SceI), and describe the recent advances in protein engineering that have led to the making of tailored meganucleases. We will then summarize the data and strategies available today regarding their use for site-directed genome modi fi cation. In the last section, we will discuss the latest data available on Transcription activator like effectors proteins as they have recently emerged as a promising new tool for genome modi fi cations.