Information on gene sequences abounds but functional genomics has not supplied the necessary information about the exact role of most of these genes. A relatively new technology, meganuclease-induced recombination, allows for highly efficient homologous gene targeting, a process where identical genes are tracked within a genome. Meganuclease-induced recombination requires highly efficient enzymes, endonucleases, that can target and cut or cleave the DNA double strand at a predefined spot. The EU-funded MEGATOOLS project therefore aimed to develop highly specific endonucleases to target one specific nucleotide stretch or DNA sequence. MEGATOOLS was a multidisciplinary project with partners who contributed the necessary input for biochemistry and molecular and cell biology as well as the technical skill for data analysis. The intention was to translate the functional genomic information into genome engineering applications that are scientifically and economically important. During the course of the project, scientists used rodent genomes to improve the technology platform for precise genome surgery. Specifically, the MEGATOOLS team successfully repaired a hamster gene sequence and incorporated genes for resistance to the drugs hygromycin, an antibiotic, and 6-thioguanine, a pharma compound used for cancer and psoriasis. The MEGATOOLS gene manipulation kits have been commercialised for target integration of gene sequences. The process involves a cut and paste action performed by the meganuclease, an integration matrix and the homologous recombination process. Meganuclease-driven targeted integration can be applied in a very wide range of biotech processes. Speeding up drug discovery is just one application. In vivo genome targeting is a highly unique and powerful tool for the functional genomics research arena.