Comprehensive analysis of the specificity of transcription activator-like effector nucleases.

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site.

A yeast-based recombination assay for homing endonuclease activity.

Homing endonucleases (HEs) are natural enzymes that cleave long DNA target with a high specificity and trigger homologous recombination at the exact site of the break. Such mechanisms can thus be used for all the applications covered today by the generic name of "genome engineering": targeted sequence insertion, removal, or editing. However, before being able to address those applications, the engineering of HEs must be mastered so that any potential target would be efficiently and specifically recognized and cleaved.

Non-specific protein-DNA interactions control I-CreI target binding and cleavage.

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.

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases.

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets.

Generation of redesigned homing endonucleases comprising DNA-binding domains derived from two different scaffolds.

Homing endonucleases have become valuable tools for genome engineering. Their sequence recognition repertoires can be expanded by modifying their specificities or by creating chimeric proteins through domain swapping between two subdomains of different homing endonucleases. Here, we show that these two approaches can be combined to create engineered meganucleases with new specificities.

Generation and analysis of mesophilic variants of the thermostable archaeal I-DmoI homing endonuclease.

The hyperthermophilic archaeon Desulfurococcus mobilis I-DmoI protein belongs to the family of proteins known as homing endonucleases (HEs). HEs are highly specific DNA-cleaving enzymes that recognize long stretches of DNA and are powerful tools for genome engineering. Because of its monomeric nature, I-DmoI is an ideal scaffold for generating mutant enzymes with novel DNA specificities, similarly reported for homodimeric HEs, but providing single chain endonucleases instead of dimers.

Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells.

Meganucleases are sequence-specific endonucleases which recognize large (>12 bp) target sites in living cells and can stimulate homologous gene targeting by a 1000-fold factor at the cleaved locus. We have recently described a combinatorial approach to redesign the I-CreI meganuclease DNA-binding interface, in order to target chosen sequences. However, engineering was limited to the protein regions shown to directly interact with DNA in a base-specific manner.

The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage.

Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein.

A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low.

Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets.

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding.

In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination.

Homing endonucleases, endonucleases capable of recognizing long DNA sequences, have been shown to be a tool of choice for precise and efficient genome engineering. Consequently, the possibility to engineer novel endonucleases with tailored specificities is under strong investigation. In this report, we present a simple and efficient method to select meganucleases from libraries of variants, based on their cleavage properties.

A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells.

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.

A revised annotation and comparative analysis of Helicobacter pylori genomes.

Huge amounts of genomic information are currently being generated. Therefore, biologists require structured, exhaustive and comparative databases. The PyloriGene database (http://genolist.