Establishing a safe harbor site for the introduction of genetic information in the human cells by the recombination system attP/attB

Resumen

The goal of this project is to generate a safe harbor site in the genome of human cells, where information could be specifically inserted without the action of nucleases. The mechanism that we present here is based on two different sitespecific recombinases phiC31 and Bxb1, as well as on the piggyBack and Sleeping Beauty transposons. Recombinase phiC31 is an integrase used by phages to establish the lysogenic life cycle. During integration, phiC31 drives recombination between the attP and the attB attachment sites on the phage and host genome, respectively. In naturally occurring phage infestations, the end result is an integrated phage genome flanked by new attL and attR sites, generated by recombination of the original, attP and attB sites. In our system, the phage genome is substituted by the acceptor sites that will constitute the core of the safe harbor locus. Under inducing conditions, the phage genome is excised via integrase-mediated recombination between attL and attR regenerating the attP and attB attachment sites. This action is directed by PhiC31 in the presence of an accessory protein (the recombination directionality factor, RDF). The alternative use of phiC31, alone or together with RDF, allows for the indefinite repetition of the cycle and the subsequent incorporation into the targeted locus of as many attachment sites as needed. The whole mechanism is made possible by the coordinated and alternative use of piggyBac and Sleeping Beauty transposons that, at each step, remove residual DNA fragments (plasmid sequences, selection elements, etc.). Once the final configuration of the safe harbor locus is reached, the Bxb1 recombinase is used to upload the desired genetic information: markers, therapeutic genes, inducible system or even complete regulatory routes.