We are characterizing novel meganucleases at the structural level. It has been shown that the I-CreI HE protein, could be extensively engineered while keeping the essential properties of HEs in terms of activity and specificity. However, as in other protein families it is quite possible that structural constraints of the I-CreI scaffold limit the scope of possible sequences that can be recognized by variants of this enzyme. Expanding the number of scaffolds could largely increase the coverage of a genomes as well as provide enzymes with better activity and/or specificity and more suitable for certain genomes. Furthermore, solving the structure of extensively redesigned I-CreI derivatives would help understand the basis of protein/DNA interaction for this family of proteins.
We are using computer aided methods to create novel meganucleases with redesigned specificity. Binding of proteins to nucleic acids is crucial for the regulation and fulfilment of many biological processes. Yet, there is no reliable method that can predict which protein will bind which nucleic acid sequence and with which affinity and specificity. The laboratory of Luis Serrano has developed FOLD-X, a computer algorithm for the structure-based prediction of the stability of proteins and protein complexes. FOLD-X is a fast computer algorithm developed by the host group for the estimation of the energetics of proteins and protein complexes from a full-atom description of their structure. Now, we are upgrading FOLD-X for the quantitative prediction of protein-nucleic acid interactions, and use it to re-engineer the meganucleases DNA binding properties.
Cellectis has created a collection of 6000 novel endonucleases derived from the I-CreI meganuclease, and methods to assemble these proteins in a combinatorial way. These studies have required the development of High Throughput Screening (HTS) methods that can now be applied to many projects. In the frame of this project, Cellectis will engineered novel proteins based on I-CreI and other scaffolds. Libraries will be created and screened on Cellectis' HTS platform, and we will try to decipher the laws and restrictions that rule the combinatorial assembly of engineered meganucleases.
Meganucleases, like restriction enzymes, perform site-specific double-stranded DNA cleavage. They recognize long ( >14 base pairs) sequences and are, therefore, extremely rare-cutting restriction enzymes. This capability makes the enzymes powerful tools in high-resolution physical mapping, analysis of genome organisation, gene cloning and site-directed induced-recombination, as well as in the studies of double-strand-break repair in diverse biological systems. However, these applications require well defined methods and high quality meganucleases that meet all quality requirements of ordinary restriction endonucleases. Therefore, a set of newly generated meganucleases will be purified and investigated by applying those procedures and high quality standards that are in use at Fermentas. These studies will provide an answer to the question if and how mutant enzymes could be used as analytical tools including for in vitro experiments.
Meganucleases have been used by many other laboratories in mammals, insects and plants , and meganuclease-induced recombination is today the most robust method to achieve gene targeting. However, experimental procedures still vary a lot depending on the organism, cell type and operator, and the locus. In order to fully validate the concept of a collection of novel meganucleases as ready-to-use reagents for genome engineering, we will use proteins produced in the course of the programme to perform genome engineering in rodent cells, and establish standard operating procedures, available for any user. Meganucleases will be used to induce homologous gene knock-out, the most common type of desired homologous gene targeting event.
