Final Report Summary - MEGATOOLS (New tools for Functional Genomics based on homologous recombination induced by double-strand break and specific meganucleases) The genome sequence programmes have contributed a huge amount of information, and opened even more possibilities. An exhaustive catalogue of genes is now available for many organisms, but the real meaning of this information remains to be deciphered. For example, most identified genes have no known function. Sometimes, homologies with other identified genes might give insight into the function, although this exercise can prove relatively perilous. In fact, a large amount of experimental work will be necessary to address the complexity of functional genomics, and the classical approaches might prove vain. Two kinds of large scale approaches have been envisioned so far. On one hand DNA chips have provided temporal information about the expression of all genes in an organism. On the other hand, knock-out and RNAi knock-down experiments provide information on the effect of loss-of-function of a particular gene. Ideally, one will like to have other possibilities like modifying, adding or subtracting genetic material in a controlled manner and target not only protein-coding sequences but any kind of DNA sequence like regulatory regions, introns etc. The way to achieve this is through meganuclease-induced recombination, a technology that allows for very highly efficient homologous gene targeting. To work properly, this approach requires enzymes that can target and cleave genes at precise locations within the chromosomes of selected cells. Very specific endonucleases should be used that ideally would target only one nucleotide stretch in a whole genome. The aim of the MEGATOOL project was to develop a large number of new sequence-specific endonucleases to recognise and target almost any possible DNA sequence in any living cell or organism, as well as to optimise homologous recombination, to provide scientists with a powerful tool to do functional genomics. In this programme, we have focussed on rodent genomes, and more specifically on the mouse genome. The MEGATOOL consortium is a partnership of academic and industrial units which provide all the necessary expertise in biochemistry, molecular-, structural-, cell-, and computational-biology to achieve its ambitious goal. In conclusion, in a research area that remains challenging and has numerous implications not only for basic research but for human health, MEGATOOLS aimed at developing new tools, significantly advancing our knowledge in homologous recombination beyond current state-of-the-art, and feeding back immediately its discoveries into the community giving it strong leverage with an enabling breakthrough, in ways that should be scientifically and economically relevant. Since meganuclease-induced recombination represents an extremely powerful tool for gene alteration, we focussed on the generation of four kinds of results:(i) A large collection of novel meganucleases This collection of novel proteins should greatly enhance the repertoire of natural meganucleases, and thus, allow for the targeting of a large number of genes in organisms whose genome has been sequenced, with a strong focus on rodent genomes.(ii) The means to exponentially increase this collection The collection of novel meganucleases should provide a unique database of characterised DNA binders. Structural and statistical studies should reveal the laws governing these interactions, and these data could in turn be used in a predictive way, for the design of novel meganucleases.(iii) The methods, procedures and quality standards to make these meganucleases widely usable as research tools.(iv) A refined method to use these meganucleases in cells The focus as on mouse cells for functional genomics, providing a direct validation.Project objectivesThe specific objectives of the project are: A) to combine state-of-the-art technologies in biochemistry, three-dimensional (3D) structure analysis and modelling, and protein engineering in order to develop target-specific meganucleases. B) to explore the development of meganucleases those are suitable for genome engineering in mouse cells.Major achievements A) Improvement of a technology platform to provide tailor-made meganucleases for the purpose of precise 'genome surgery'; B) Crystallisation and structure resolution of several meganucleases in complex with their cognate target; C) Improvement of the computer algorithm FOLD-X for quantitative prediction of protein-DNA interactions; D) Implementation of the Megatoolbase; E) Production of custom-made meganucleases targeting the ROSA26, Glutamate synthetase (GS), Hamster's Hypoxanthine ribosyl transferase (HPRT) mouse genes; F) Establishment of standard procedure for genome engineering; G) Commercialisation of the first meganuclease-based genome engineering tools. Publishable resultsMeganucleases can be engineered to change their recognition site sequence. This breakthrough in protein engineering alleviates the need to pre-engineer cell lines with a natural meganuclease recognition site. Engineered meganucleases achieve targeted integration on 'wild type' cell lines.The first ever engineered meganuclease for the hamster genome targets the natural Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. HPRT is involved in purine metabolism and converts 6-thioguanine (6-TG) to its monophosphate form (6-TGMP). 6-TGMP interferes subsequently with de novo synthesis of purines.CHO-K1 HPRT+ cells are naturally sensitive to 6'TG. Upon expression of the engineered meganuclease targeting HPRT following cell transfection, a DNA double-strand break is made which triggers repair through homologous recombination. The transfected integration matrix is used for repair and contains upstream and downstream homology regions of the HPRT locus, a hygromycin resistance gene, and promoter and terminator sequences of the Gene of interest (GOI) coding sequence. Upon repair, the GOI is integrated at the HPRT meganuclease recognition site, and the cells become hygromycin and 6-TG resistant (HPRT deficient). Related documents Final Report - MEGATOOLS (New tools for Functional Genomics based on homologous recombination induced by double-strand break and specific meganucleases)
